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Journal of Pharmaceutical and Biomedical Analysis 129 (2016) 28–33
Contents lists available at ScienceDirect
Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba
Short communication
Pharmacokinetic profile of cefbuperazone in healthy Chinese volunteers after single and multiple drip intravenous infusion by HPLC–MS/MS Dongbo Liu a,b , Taohua Geng a,b , Yiya Wang b,∗∗ , Li Ding a,b,∗ a b
Department of Pharmaceutical Analysis, China Pharmaceutical University, Nanjing 210009, China Nanjing Clinical Tech. Laboratories Inc., Nanjing 211100, China
a r t i c l e
i n f o
Article history: Received 20 January 2016 Received in revised form 3 June 2016 Accepted 19 June 2016 Available online 23 June 2016 Keywords: Cefbuperazone HPLC–MS/MS Pharmacokinetic Excretion
a b s t r a c t A selective and reproducible HPLC–MS/MS method was developed and fully validated for the determination of cefbuperazone in human plasma and urine. Samples were prepared using protein precipitation and separated on a Zorbax Eclipse Plus C18 column (2.1 × 50 mm, 3.5 m). The API-4000 mass spectrometer was operated under multiple reaction monitoring mode (MRM) using the electrospray ionization technique. Linearity was achieved from 0.250 to 250 g/mL in plasma and 20.0–5000 g/mL in urine. The method was successfully applied to a pharmacokinetic study of cefbuperazone in healthy Chinese volunteers after drip intravenous infusion of 0.5, 1.0, 2.0 g cefbuperazone sodium injection. Cefbuperazone reached a maximum concentration (Cmax ) of 44.7 ± 8.1 g/mL, 86.7 ± 12.7 g/mL and 168 ± 14 g/mL in 0.5, 1.0 and 2.0 g dose groups respectively, at 60 min after the start of infusion. The half-life (t1/2 ) was between 1.8–1.9 h, and the elimination constant (kel ) was between 0.36–0.39 h−1 . The results proved that cefbuperazone showed linear pharmacokinetic profile in the dose range of 0.5–2.0 g without gender difference. Drug accumulation was not observed. Cefbuperazone reached the maximum excretion rate in urine 2 h after the start of infusion. About 60.0% of the administered drug was excreted via urine as unchanged form within 12 h. The cumulative excretion of cefbuperazone after single drip intravenous infusion was proportional to the administered dose within the range from 0.5 g to 2.0 g. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Cefbuperazone, developed by Toyama Chemical Co., Ltd, is an antibiotic belonging to the second generation of cephamycin. It shows a strong antibacterial activity especially against the Gram-negative rod and anaerobic bacteria [1,2]. According to the manufacturer’s instruction, the action mechanism of cefbuperazone is the inhibition of the bacterial cell wall synthesis. Since cefbuperazone is excellent in permeability of bacterial outer membrane and exerts its bactericidal activity by strongly binding to penicillin-binding protein (PBP) 1A, 1B, and 3s, bacterial regrowth
Abbreviations: IS, internal standard; LLOQ, lower limit of quantification; ULOQ, upper limit of quantification; LQC, low quality control; HQC, high quality control; RSD, relative standard deviation. ∗ Corresponding author at: Department of Pharmaceutical Analysis, China Pharmaceutical University, Nanjing 210009, China. ∗∗ Corresponding author. E-mail addresses: [email protected] (Y. Wang), [email protected] (L. Ding). http://dx.doi.org/10.1016/j.jpba.2016.06.029 0731-7085/© 2016 Elsevier B.V. All rights reserved.
cannot take place easily. This study aimed to identify the pharmacokinetic profile of cefbuperazone after single and multiple drip intravenous infusion in healthy Chinese subjects, and to evaluate the drug accumulation.
2. Experimental 2.1. Chemicals and reagents The reference standard, cefbuperazone sodium was obtained from Salubris Pharmaceutical Co., Ltd. (Shenzhen, China). The internal standard (IS), tinidazole, was purchased from National Institutes for Food and Drug Control (Beijing, China). HPLC grade methanol and acetonitrile was purchased from Merck KGaA (Darmstadt, Germany). Deionized water was freshly prepared by a water purification system (Milli-Q Reference, Millipore, Boston, MA, USA). Analytical grade acetic acid and ammonium acetate were obtained from Sigma-Aldrich (St. Louis, MO, USA).
D. Liu et al. / Journal of Pharmaceutical and Biomedical Analysis 129 (2016) 28–33
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Fig. 1. The typical MRM chromatograms: (A) blank plasma sample; (B) blank plasma sample spiked with the analyte at LLOQ concentration and the IS; (C) a plasma sample obtained 75 min from the start of infusion of 1.0 g cefbuperazone sodium injection in single-dose study.
2.2. Sample preparation An aliquot of 50 L of the IS working solution (2.00 g/mL) and 200 L acetonitrile was added to 50 L of plasma sample in a 96well plate. Then, the solution was thoroughly vortex-mixed for 10 min. After centrifugation at 4000 rpm, 25 ◦ C for 10 min, 30 L of the supernatant was transferred into a clean 96-well plate containing 570 L deionized water. The plate was placed into auto-sampler at 8 ◦ C after vortexing for 10 min. Each urine sample (100 L) was added with 100 L of the IS working solution (20.0 g/mL) and 400 L of acetonitrile. After thoroughly vortex-mixing and centrifuging at 4000 rpm, 25 ◦ C for 5 min, 30 L of the supernatant was diluted with 570 L deionized water and vortexed for 10 min. Then 30 L of this solution was mixed with 570 L deionized water again. 5 L of the solution was injected for analysis. 2.3. Preparation of calibration standards and quality control samples Cefbuperazone sodium stock solutions for calibration standards and quality control (QC) samples were prepared from separately weight. The stock solution was prepared in 50% acetonitrile and stored at –20 ◦ C. Calibration standards covered the concentration levels of 0.250, 0.500, 2.00, 10.0, 50.0, 100, 200 and 250 g/mL.
QC samples were prepared at 0.600, 20.0, 80.0 and 190 g/mL. For urine samples, the concentrations of calibration standards were of 20, 40, 150, 600, 2000, 3500 and 5000 g/mL. QC samples were prepared at 50, 1000 and 4000 g/mL. 2.4. HPLC–MS/MS instruments and conditions Chromatographic separation was performed on a Zorbax Eclipse Plus C18 column (2.1 × 50 mm, 3.5 m) with an HPLC system (Shimadzu, Kyoto, Japan). Two elution techniques were used for the determination of cefbuperazone in plasma and urine samples. Gradient elution was applied with 5 mM ammonium acetate-formic acid (100:0.1, v/v, mobile phase A) and acetonitrile (mobile phase B). The following gradient program was used for plasma separation: 0–2.4 min, 14% B; 2.4–2.6 min, 14 → 90% B; 2.6–3.6 min, 90% B; 3.6–3.8 min, 90 → 14% B; 3.8–5.5 min, 14% B. In the other hand, an isocratic mobile phase consisting of 14% B in 3 min was used for urine samples. The mobile phase was delivered at 0.35 mL/min. The MS/MS was performed on an Applied Biosystems/Sciex API-4000 mass spectrometer (Sciex, Framingham, MA, USA) under positiveion multiple reaction monitoring mode (MRM). The transition pairs were at m/z 628.1 → 512.1 for cefbuperazone and 248.1 → 121.1 for tinidazole. The analytical data were acquired and processed by Analyst software (Version 1.6.2, Sciex).
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D. Liu et al. / Journal of Pharmaceutical and Biomedical Analysis 129 (2016) 28–33
Fig. 2. The typical MRM chromatograms: (A) blank urine sample; (B) blank urine sample spiked with the analyte at LLOQ concentration and the IS; (C) a urine sample obtained post-dose in the time intervals of 0–2 h of 0.5 g cefbuperazone sodium injection in single-dose study.
2.5. Pharmacokinetic study A total of 36 healthy Chinese volunteers with age ranging from 19 to 29 (24 ± 3, mean ± SD) and body mass index (BMI) ranging from 19.0 to 24.2 kg/m2 (21.4 ± 1.7 kg/m2 , mean ± SD) participated in the study after signing the informed consent. The protocol was obedient to the ethical principles established in the Declaration of Helsinki and approved by the Ethics Committee of the First Affiliated Hospital of Suzhou University (Suzhou, China). All subjects underwent physical examinations, electrocardiograms and routine laboratory tests before and after the study. 2.5.1. Single-dose administration All volunteers were randomly divided into three dose groups (0.5, 1.0 and 2.0 g). Each subject fasted for at least 10 h and served with light breakfast before drip intravenous infusion at constant speed for 1 h. Blood samples were collected into heparinized tubes prior to dosing and 20 min, 40 min, 60 min (drip infusion stop), 65 min, 75 min, 90 min, 2 h, 2.5 h, 3 h, 4 h, 6 h, 8 h and 12 h from the start of infusion. Plasma was harvested after centrifuged (3500 rpm for 10 min at 4 ◦ C) and mixed with acetic acid (100:1, v/v), and then stored at −65 ◦ C immediately until analysis. Urine samples were obtained prior to dose and post-dose in the time intervals of 0–2 h, 2–4 h, 4–6 h, 6–8 h and 8–12 h. At the end of a given time
interval, 10 mL of the urine was transferred into a labeled tube containing acetic acid (100:1, v/v) and thoroughly vortex-mixed. Urine samples were kept frozen at –20 ◦ C until analysis.
2.5.2. Multiple-doses administration After single-dose administration, 12 volunteers of the middle dose group (1.0 g) were treated with 1.0 g cefbuperazone sodium injections for 5 consecutive days at 12 h interval. On days 3, 4 and 5, venous blood before every administration was drawn to quantify the trough plasma concentration. After steady state had been reached on day 5, plasma samples were obtained at the same time points as the single-dose administration.
2.5.3. Data analysis The maximum concentration (Cmax ) and time of reaching maximum concentration (Tmax ) were obtained from a concentration–time profile. Non-compartmental pharmacokinetic analysis was performed using DAS software (Version 3.2, Chinese Pharmacology Society, Beijing, China) to calculate PK parameters. Statistical analysis was conducted with SPSS software (Version 19.0, IBM, Armonk, NY, USA).
D. Liu et al. / Journal of Pharmaceutical and Biomedical Analysis 129 (2016) 28–33
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Fig. 3. Mean plasma concentration-time profiles of cefbuperazone in healthy Chinese volunteers (n = 12 per dose) after single and multiple drip intravenous infusion of cefbuperazone sodium injection.
3. Results and discussion
stance interference was observed. Linearity was established in the concentration range of 0.250–250 g/mL for plasma and of 20.0–5000 g/mL for urine. The LLOQs were 0.250 and 20.0 g/mL for plasma and urine respectively, which were judged from the fact that the precision and accuracy were less than 20% and the signalto-noise ratios (S/N) were much higher than 10. The typical MRM chromatograms are presented in Fig. 1 and 2.
3.1. Method optimization In sample preparation procedure, a variety of precipitants were investigated. Acetonitrile showed good recovery without matrix effect. Quantitative acetic acid was added into samples immediately to avoid the hydrolysis of -lactam structure. When selecting the mobile phase attention was paid to the influence on the chromatographic peak shape and retention time. Acetonitrile was found superior to methanol to decrease column pressure and lower background noise. The chromatographic peak became sharper and more symmetrical with the use of 5 mM ammonium acetate buffer. The addition of 0.1% formic acid was performed to obtain higher response. Compared with previous methods [3,4], this HPLC–MS/MS method had lower limit of detection with high throughput and shorter run time. The required sample volume was much less.
3.2.2. Accuracy and precision For plasma samples, the RSD values of intra-day precision and inter-day precision were within 7.0%. The accuracy at five QC levels (n = 6) ranged from 98.1% to 106.0%. The method for urine samples had satisfactory reproducibility with accuracy ranging from 101.4% to 114.0%. The precision values were less than 11.4% at four QC levels (n = 6). 3.2.3. Recovery, matrix effect and carry-over The extraction recoveries were (91.0 ± 1.2)% and (94.8 ± 0.2)% for plasma and urine respectively. The matrix effects of cefbuperazone after normalization with the IS, were (102.1 ± 0.7)% and (101.4 ± 1.4)% for plasma and urine respectively. Carry-over was evaluated by injecting blank sample after ULOQ to determine the interference at the retention time of the analyte and the IS. No carry-over was observed.
3.2. Methods validation 3.2.1. Specificity, linearity and LLOQs Specificity was determined by analysis of blank human plasma and urine samples from six different sources. No endogenous sub-
Table 1 Pharmacokinetic parameters of cefbuperazone in healthy Chinese volunteers (n = 12 per dose) after single and multiple drip intravenous infusion of cefbuperazone sodium injection. Parameters
Cmax (g mL−1 ) Tmax (h) t1/2 (h) AUC0−12 (g h mL−1 ) AUC0–∞ (g h mL−1 ) kel (h−1 ) Vd /F (L) CL/F (L/h) MRT0−12 (h) MRT0–∞ (h) Cav (g mL−1 ) Cssmin (g mL−1 ) Cssmax (g mL−1 ) AUCss (g h mL−1 ) DF
0.5 g
1.0 g
Single dose
Single dose
Multiple dose
Single dose
44.7 ± 8.1 1 1.9 ± 0.1 114 ± 22 116 ± 22 0.36 ± 0.03 12.4 ± 2.3 4.5 ± 0.8 2.7 ± 0.1 2.9 ± 0.2 – – – – –
86.7 ± 12.7 1 1.8 ± 0.3 221 ± 35 224 ± 13 0.39 ± 0.06 11.9 ± 1.8 4.6 ± 0.7 2.7 ± 0.3 2.9 ± 0.4 – – – – –
94.9 ± 12.8 1 1.9 ± 0.2 237 ± 34 240 ± 35 0.38 ± 0.04 11.3 ± 1.7 4.2 ± 0.7 2.7 ± 0.2 2.8 ± 0.3 19.8 ± 2.9 1.6 ± 0.7 94.9 ± 12.8 238 ± 34 4.7 ± 0.5
168 ± 14 1 1.9 ± 0.2 422 ± 58 427 ± 62 0.37 ± 0.04 12.8 ± 1.1 4.8 ± 0.7 2.7 ± 0.2 2.8 ± 0.3 – – – – –
2.0 g
Cav , the average concentration of steady state; Css min , the minimum value of the steady plasma drug concentration; Css max , the maximum value of the steady plasma drug concentration; AUCss, area under the plasma concentration-time curve of the steady state; DF, degree of fluctuation.
32
D. Liu et al. / Journal of Pharmaceutical and Biomedical Analysis 129 (2016) 28–33
Fig. 4. Mean urinary cumulative excretion amount-time profiles of cefbuperazone in healthy Chinese volunteers (n = 12 per dose) after single drip intravenous infusion of cefbuperazone sodium injection.
3.2.4. Stability For plasma samples, the short-term stability was determined with QC samples containing 1% acetic acid (v/v) stored for 16 h and QC samples without acetic acid stored for 2.9 h at room temperature. The freeze-thaw stability was examined at five freeze-thaw cycles between –65/−20◦C and room temperature. The longterm stability was tested after storage of acidulated samples at −65/–20 ◦ C for 62 days. The post-exacted stability was assessed with left QC samples in the auto-sampler for 87 h at 8 ◦ C. For urine samples, the short-term stability was measured with QC samples containing 1% acetic acid (v/v) stored for 16 h at room temperature. The urine samples without acetic acid were stored at room temperature for 2.2 h and at 2–8 ◦ C for 5.4 h. The freeze-thaw stability was examined at five freeze-thaw cycles between −20 ◦ C and room temperature. The long-term stability was determined after storage of acidulated urine samples at −20 ◦ C for 35 days. The post-exacted stability was assessed with left QC samples in the auto-sampler for 48 h at 8 ◦ C. Stability study demonstrated no significant changes in QC samples.
ceedings [5,6] and the reported studies [7,8], our PK parameters are obtained from healthy Chinese volunteers. However, the PK parameters in existing literature are obtained from patients. 3.3.2. Multiple-dose study The trough plasma concentrations from day 3–5 showed no significant difference (P > 0.05, one-way ANOVA), indicating that steady state had been reached after consecutive administration for 3 days at a 12-h interval. The PK parameters are summarized in Tab 1. Compared with the single-dose study, the main PK parameters showed no significant difference. The accumulation index (RAUC ) was (1.08 ± 0.04), proving that there was no drug accumulation tendency in healthy Chinese subjects.
3.3. Pharmacokinetic results
3.3.3. Gender effects The PK parameters after single-dose and multiple-dose administration are divided by gender in Tab 2. The Cmax and AUC0–12h were corrected with dose and weight. The main PK parameters showed no significant difference between two gender groups (P > 0.05, independent-samples t-test), indicating that PK behavior of cefbuperazone had no gender difference.
3.3.1. Single-dose study Following drip intravenous infusion, plasma levels of cefbuperazone reached maximum at 60 min and rapidly eliminated. Mean plasma concentration-time profiles after single-dose administration are presented in Fig. 3. The PK parameters are reported in Table 1. No significant difference was identified in pharmacokinetic parameters of t1/2 , Vd/F, CL/F and kel between the three dose groups. There was an excellent linear relationship between AUC0−12h and dose, as well as Cmax and dose. Compared with the conference pro-
3.3.4. Urinary excretion Mean urinary cumulative excretion amount-time profiles are shown in Fig. 4. The concentration of cefbuperazone in urine samples collected during 0–2 h was highest and then decreased rapidly. About 60.0% of the administered drug was excreted via urine as unchanged form within 12 h, indicating that the dominant elimination pathway for cefbuperazone in healthy Chinese subjects was urinary excretion. Dose-normalized cumulative excretion amount showed no significant difference between the three dose groups
Table 2 Pharmacokinetic parameters of cefbuperazone in healthy Chinese volunteers (n = 12 per dose) divided by gender after single and multiple drip intravenous infusion of cefbuperazone sodium injection. 1.0 g 0.5 g Parameters −1
Cmax (g mL ) Tmax (h) AUC0–12 (g h mL-1 ) t1/2 (h) MRT0 − 12 (h) CL/F (L/h) Vd /F (L) AUCss (g h mL−1 ) Cav (g mL−1 )
Single dose
Multiple dose
2.0 g
female
male
female
male
female
male
female
male
42.4 ± 6.6 1 109 ± 20 2.0 ± 0.1 2.7 ± 0.1 4.7 ± 0.9 13.1 ± 2.4 – –
47.1 ± 9.3 1 120 ± 23 1.9 ± 0.2 2.7 ± 0.2 4.3 ± 0.8 11.8 ± 2.1 – –
93.7 ± 14.8 1 227 ± 42 1.7 ± 0.2 2.6 ± 0.2 4.5 ± 0.9 10.8 ± 1.6 – –
79.7 ± 4.3 1 215 ± 28 2.0 ± 0.2 2.9 ± 0.2 4.6 ± 0.6 13.1 ± 1.1 – –
101 ± 16 1 243 ± 46 1.7 ± 0.1 2.5 ± 0.2 4.2 ± 0.9 10.5 ± 2.0 242.9 ± 45.6 20.2 ± 3.8
89.0 ± 3.8 1 232 ± 21 2.0 ± 0.2 2.8 ± 0.2 4.3 ± 0.4 12.0 ± 1.1 232.1 ± 21.4 19.3
174 ± 14 1 425 ± 54 1.9 ± 0.2 2.7 ± 0.2 4.7 ± 0.6 12.5 ± 1.4 – –
162 ± 11 1 418 ± 68 1.9 ± 0.2 2.7 ± 0.3 4.8 ± 0.8 13.0 ± 0.7 – –
D. Liu et al. / Journal of Pharmaceutical and Biomedical Analysis 129 (2016) 28–33
(P > 0.05, one-way ANOVA), proving that urinary cumulative excretion was proportional to the administered dose within the range of 0.5–2.0 g. 4. Conclusion A selective and reproducible HPLC–MS/MS method for the determination of cefbuperazone in human plasma and urine was established, validated and successfully applied to a pharmacokinetic study. The pharmacokinetic profile of cefbuperazone and accumulation in healthy Chinese subjects were studied in detail. Cefbuperazone has a dose-independent pharmacokinetic profile without drug accumulation tendency. The dominant elimination pathway was urinary excretion as unchanged form. References [1] F.Z. Jia, Clinical application of cephamycin antibiotics, Pract. Geriatr. 12 (1998) 169–170. [2] J.M.T. Hamilton-Miller, W. Brumfitt, Cephamycins: a review, prospects and some original observations, Infection 3 (1975) 183–188.
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[3] H.K. Mei, N. Bai, B.B. Liang, Y. Cai, R. Wang, Determination concentration of cefbuperazone in human plasma and urine by HPLC, Chin. J. Clin. Pharmacol. 28 (2012) 56–58. [4] Y.Y. Han, The Study of Pharmacokinetics and Pharmacodynamics of Four Kinds of Bacterial of Cefbuperazone Sodium, Hebei North University, Hebei, 2012, pp. 9–13. [5] Y. Akieda, I. Nakayama, H. Kawamura, H. Kawaguchi, E. Yamaji, Comparative pharmacokinetic studies of cefotetan and other cephamycin antibiotics: cefminox, cefbuperazone, cefmetazole, cefoxitin, and latamoxef, Recent Adv Chemother. Proc. Int. Congr. Chemother., 14th, University of Tokyo Press, (1985) 1111–1112. [6] M. Takefumi, Y. Yamada, J. Sasaki, H. Uematsu, H. Sakamoto, K. Shiiki, Distribution and pharmacokinetics of cefbuperazone in human and New Zealand white (NZW) rabbits, Proc. int. congr. chemother. 13th Verlag H. Egermann, (1983) 64–66. [7] K. Seiga, Y. Sugiyama, Pharmacokinetic studies of cefbuperazone in the field of gynecology, Jpn. J. Antibiot. 37 (1984) 2519–2530. [8] Y. Watanabe, A. Gonda, T. Kidokoro, Penetration of cefbuperazone a new cephamycin antibiotic, into human peritoneal exudate, Jpn. J. Antibiot. 37 (1984) 261–266.
Contents lists available at ScienceDirect
Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba
Short communication
Pharmacokinetic profile of cefbuperazone in healthy Chinese volunteers after single and multiple drip intravenous infusion by HPLC–MS/MS Dongbo Liu a,b , Taohua Geng a,b , Yiya Wang b,∗∗ , Li Ding a,b,∗ a b
Department of Pharmaceutical Analysis, China Pharmaceutical University, Nanjing 210009, China Nanjing Clinical Tech. Laboratories Inc., Nanjing 211100, China
a r t i c l e
i n f o
Article history: Received 20 January 2016 Received in revised form 3 June 2016 Accepted 19 June 2016 Available online 23 June 2016 Keywords: Cefbuperazone HPLC–MS/MS Pharmacokinetic Excretion
a b s t r a c t A selective and reproducible HPLC–MS/MS method was developed and fully validated for the determination of cefbuperazone in human plasma and urine. Samples were prepared using protein precipitation and separated on a Zorbax Eclipse Plus C18 column (2.1 × 50 mm, 3.5 m). The API-4000 mass spectrometer was operated under multiple reaction monitoring mode (MRM) using the electrospray ionization technique. Linearity was achieved from 0.250 to 250 g/mL in plasma and 20.0–5000 g/mL in urine. The method was successfully applied to a pharmacokinetic study of cefbuperazone in healthy Chinese volunteers after drip intravenous infusion of 0.5, 1.0, 2.0 g cefbuperazone sodium injection. Cefbuperazone reached a maximum concentration (Cmax ) of 44.7 ± 8.1 g/mL, 86.7 ± 12.7 g/mL and 168 ± 14 g/mL in 0.5, 1.0 and 2.0 g dose groups respectively, at 60 min after the start of infusion. The half-life (t1/2 ) was between 1.8–1.9 h, and the elimination constant (kel ) was between 0.36–0.39 h−1 . The results proved that cefbuperazone showed linear pharmacokinetic profile in the dose range of 0.5–2.0 g without gender difference. Drug accumulation was not observed. Cefbuperazone reached the maximum excretion rate in urine 2 h after the start of infusion. About 60.0% of the administered drug was excreted via urine as unchanged form within 12 h. The cumulative excretion of cefbuperazone after single drip intravenous infusion was proportional to the administered dose within the range from 0.5 g to 2.0 g. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Cefbuperazone, developed by Toyama Chemical Co., Ltd, is an antibiotic belonging to the second generation of cephamycin. It shows a strong antibacterial activity especially against the Gram-negative rod and anaerobic bacteria [1,2]. According to the manufacturer’s instruction, the action mechanism of cefbuperazone is the inhibition of the bacterial cell wall synthesis. Since cefbuperazone is excellent in permeability of bacterial outer membrane and exerts its bactericidal activity by strongly binding to penicillin-binding protein (PBP) 1A, 1B, and 3s, bacterial regrowth
Abbreviations: IS, internal standard; LLOQ, lower limit of quantification; ULOQ, upper limit of quantification; LQC, low quality control; HQC, high quality control; RSD, relative standard deviation. ∗ Corresponding author at: Department of Pharmaceutical Analysis, China Pharmaceutical University, Nanjing 210009, China. ∗∗ Corresponding author. E-mail addresses: [email protected] (Y. Wang), [email protected] (L. Ding). http://dx.doi.org/10.1016/j.jpba.2016.06.029 0731-7085/© 2016 Elsevier B.V. All rights reserved.
cannot take place easily. This study aimed to identify the pharmacokinetic profile of cefbuperazone after single and multiple drip intravenous infusion in healthy Chinese subjects, and to evaluate the drug accumulation.
2. Experimental 2.1. Chemicals and reagents The reference standard, cefbuperazone sodium was obtained from Salubris Pharmaceutical Co., Ltd. (Shenzhen, China). The internal standard (IS), tinidazole, was purchased from National Institutes for Food and Drug Control (Beijing, China). HPLC grade methanol and acetonitrile was purchased from Merck KGaA (Darmstadt, Germany). Deionized water was freshly prepared by a water purification system (Milli-Q Reference, Millipore, Boston, MA, USA). Analytical grade acetic acid and ammonium acetate were obtained from Sigma-Aldrich (St. Louis, MO, USA).
D. Liu et al. / Journal of Pharmaceutical and Biomedical Analysis 129 (2016) 28–33
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Fig. 1. The typical MRM chromatograms: (A) blank plasma sample; (B) blank plasma sample spiked with the analyte at LLOQ concentration and the IS; (C) a plasma sample obtained 75 min from the start of infusion of 1.0 g cefbuperazone sodium injection in single-dose study.
2.2. Sample preparation An aliquot of 50 L of the IS working solution (2.00 g/mL) and 200 L acetonitrile was added to 50 L of plasma sample in a 96well plate. Then, the solution was thoroughly vortex-mixed for 10 min. After centrifugation at 4000 rpm, 25 ◦ C for 10 min, 30 L of the supernatant was transferred into a clean 96-well plate containing 570 L deionized water. The plate was placed into auto-sampler at 8 ◦ C after vortexing for 10 min. Each urine sample (100 L) was added with 100 L of the IS working solution (20.0 g/mL) and 400 L of acetonitrile. After thoroughly vortex-mixing and centrifuging at 4000 rpm, 25 ◦ C for 5 min, 30 L of the supernatant was diluted with 570 L deionized water and vortexed for 10 min. Then 30 L of this solution was mixed with 570 L deionized water again. 5 L of the solution was injected for analysis. 2.3. Preparation of calibration standards and quality control samples Cefbuperazone sodium stock solutions for calibration standards and quality control (QC) samples were prepared from separately weight. The stock solution was prepared in 50% acetonitrile and stored at –20 ◦ C. Calibration standards covered the concentration levels of 0.250, 0.500, 2.00, 10.0, 50.0, 100, 200 and 250 g/mL.
QC samples were prepared at 0.600, 20.0, 80.0 and 190 g/mL. For urine samples, the concentrations of calibration standards were of 20, 40, 150, 600, 2000, 3500 and 5000 g/mL. QC samples were prepared at 50, 1000 and 4000 g/mL. 2.4. HPLC–MS/MS instruments and conditions Chromatographic separation was performed on a Zorbax Eclipse Plus C18 column (2.1 × 50 mm, 3.5 m) with an HPLC system (Shimadzu, Kyoto, Japan). Two elution techniques were used for the determination of cefbuperazone in plasma and urine samples. Gradient elution was applied with 5 mM ammonium acetate-formic acid (100:0.1, v/v, mobile phase A) and acetonitrile (mobile phase B). The following gradient program was used for plasma separation: 0–2.4 min, 14% B; 2.4–2.6 min, 14 → 90% B; 2.6–3.6 min, 90% B; 3.6–3.8 min, 90 → 14% B; 3.8–5.5 min, 14% B. In the other hand, an isocratic mobile phase consisting of 14% B in 3 min was used for urine samples. The mobile phase was delivered at 0.35 mL/min. The MS/MS was performed on an Applied Biosystems/Sciex API-4000 mass spectrometer (Sciex, Framingham, MA, USA) under positiveion multiple reaction monitoring mode (MRM). The transition pairs were at m/z 628.1 → 512.1 for cefbuperazone and 248.1 → 121.1 for tinidazole. The analytical data were acquired and processed by Analyst software (Version 1.6.2, Sciex).
30
D. Liu et al. / Journal of Pharmaceutical and Biomedical Analysis 129 (2016) 28–33
Fig. 2. The typical MRM chromatograms: (A) blank urine sample; (B) blank urine sample spiked with the analyte at LLOQ concentration and the IS; (C) a urine sample obtained post-dose in the time intervals of 0–2 h of 0.5 g cefbuperazone sodium injection in single-dose study.
2.5. Pharmacokinetic study A total of 36 healthy Chinese volunteers with age ranging from 19 to 29 (24 ± 3, mean ± SD) and body mass index (BMI) ranging from 19.0 to 24.2 kg/m2 (21.4 ± 1.7 kg/m2 , mean ± SD) participated in the study after signing the informed consent. The protocol was obedient to the ethical principles established in the Declaration of Helsinki and approved by the Ethics Committee of the First Affiliated Hospital of Suzhou University (Suzhou, China). All subjects underwent physical examinations, electrocardiograms and routine laboratory tests before and after the study. 2.5.1. Single-dose administration All volunteers were randomly divided into three dose groups (0.5, 1.0 and 2.0 g). Each subject fasted for at least 10 h and served with light breakfast before drip intravenous infusion at constant speed for 1 h. Blood samples were collected into heparinized tubes prior to dosing and 20 min, 40 min, 60 min (drip infusion stop), 65 min, 75 min, 90 min, 2 h, 2.5 h, 3 h, 4 h, 6 h, 8 h and 12 h from the start of infusion. Plasma was harvested after centrifuged (3500 rpm for 10 min at 4 ◦ C) and mixed with acetic acid (100:1, v/v), and then stored at −65 ◦ C immediately until analysis. Urine samples were obtained prior to dose and post-dose in the time intervals of 0–2 h, 2–4 h, 4–6 h, 6–8 h and 8–12 h. At the end of a given time
interval, 10 mL of the urine was transferred into a labeled tube containing acetic acid (100:1, v/v) and thoroughly vortex-mixed. Urine samples were kept frozen at –20 ◦ C until analysis.
2.5.2. Multiple-doses administration After single-dose administration, 12 volunteers of the middle dose group (1.0 g) were treated with 1.0 g cefbuperazone sodium injections for 5 consecutive days at 12 h interval. On days 3, 4 and 5, venous blood before every administration was drawn to quantify the trough plasma concentration. After steady state had been reached on day 5, plasma samples were obtained at the same time points as the single-dose administration.
2.5.3. Data analysis The maximum concentration (Cmax ) and time of reaching maximum concentration (Tmax ) were obtained from a concentration–time profile. Non-compartmental pharmacokinetic analysis was performed using DAS software (Version 3.2, Chinese Pharmacology Society, Beijing, China) to calculate PK parameters. Statistical analysis was conducted with SPSS software (Version 19.0, IBM, Armonk, NY, USA).
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Fig. 3. Mean plasma concentration-time profiles of cefbuperazone in healthy Chinese volunteers (n = 12 per dose) after single and multiple drip intravenous infusion of cefbuperazone sodium injection.
3. Results and discussion
stance interference was observed. Linearity was established in the concentration range of 0.250–250 g/mL for plasma and of 20.0–5000 g/mL for urine. The LLOQs were 0.250 and 20.0 g/mL for plasma and urine respectively, which were judged from the fact that the precision and accuracy were less than 20% and the signalto-noise ratios (S/N) were much higher than 10. The typical MRM chromatograms are presented in Fig. 1 and 2.
3.1. Method optimization In sample preparation procedure, a variety of precipitants were investigated. Acetonitrile showed good recovery without matrix effect. Quantitative acetic acid was added into samples immediately to avoid the hydrolysis of -lactam structure. When selecting the mobile phase attention was paid to the influence on the chromatographic peak shape and retention time. Acetonitrile was found superior to methanol to decrease column pressure and lower background noise. The chromatographic peak became sharper and more symmetrical with the use of 5 mM ammonium acetate buffer. The addition of 0.1% formic acid was performed to obtain higher response. Compared with previous methods [3,4], this HPLC–MS/MS method had lower limit of detection with high throughput and shorter run time. The required sample volume was much less.
3.2.2. Accuracy and precision For plasma samples, the RSD values of intra-day precision and inter-day precision were within 7.0%. The accuracy at five QC levels (n = 6) ranged from 98.1% to 106.0%. The method for urine samples had satisfactory reproducibility with accuracy ranging from 101.4% to 114.0%. The precision values were less than 11.4% at four QC levels (n = 6). 3.2.3. Recovery, matrix effect and carry-over The extraction recoveries were (91.0 ± 1.2)% and (94.8 ± 0.2)% for plasma and urine respectively. The matrix effects of cefbuperazone after normalization with the IS, were (102.1 ± 0.7)% and (101.4 ± 1.4)% for plasma and urine respectively. Carry-over was evaluated by injecting blank sample after ULOQ to determine the interference at the retention time of the analyte and the IS. No carry-over was observed.
3.2. Methods validation 3.2.1. Specificity, linearity and LLOQs Specificity was determined by analysis of blank human plasma and urine samples from six different sources. No endogenous sub-
Table 1 Pharmacokinetic parameters of cefbuperazone in healthy Chinese volunteers (n = 12 per dose) after single and multiple drip intravenous infusion of cefbuperazone sodium injection. Parameters
Cmax (g mL−1 ) Tmax (h) t1/2 (h) AUC0−12 (g h mL−1 ) AUC0–∞ (g h mL−1 ) kel (h−1 ) Vd /F (L) CL/F (L/h) MRT0−12 (h) MRT0–∞ (h) Cav (g mL−1 ) Cssmin (g mL−1 ) Cssmax (g mL−1 ) AUCss (g h mL−1 ) DF
0.5 g
1.0 g
Single dose
Single dose
Multiple dose
Single dose
44.7 ± 8.1 1 1.9 ± 0.1 114 ± 22 116 ± 22 0.36 ± 0.03 12.4 ± 2.3 4.5 ± 0.8 2.7 ± 0.1 2.9 ± 0.2 – – – – –
86.7 ± 12.7 1 1.8 ± 0.3 221 ± 35 224 ± 13 0.39 ± 0.06 11.9 ± 1.8 4.6 ± 0.7 2.7 ± 0.3 2.9 ± 0.4 – – – – –
94.9 ± 12.8 1 1.9 ± 0.2 237 ± 34 240 ± 35 0.38 ± 0.04 11.3 ± 1.7 4.2 ± 0.7 2.7 ± 0.2 2.8 ± 0.3 19.8 ± 2.9 1.6 ± 0.7 94.9 ± 12.8 238 ± 34 4.7 ± 0.5
168 ± 14 1 1.9 ± 0.2 422 ± 58 427 ± 62 0.37 ± 0.04 12.8 ± 1.1 4.8 ± 0.7 2.7 ± 0.2 2.8 ± 0.3 – – – – –
2.0 g
Cav , the average concentration of steady state; Css min , the minimum value of the steady plasma drug concentration; Css max , the maximum value of the steady plasma drug concentration; AUCss, area under the plasma concentration-time curve of the steady state; DF, degree of fluctuation.
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Fig. 4. Mean urinary cumulative excretion amount-time profiles of cefbuperazone in healthy Chinese volunteers (n = 12 per dose) after single drip intravenous infusion of cefbuperazone sodium injection.
3.2.4. Stability For plasma samples, the short-term stability was determined with QC samples containing 1% acetic acid (v/v) stored for 16 h and QC samples without acetic acid stored for 2.9 h at room temperature. The freeze-thaw stability was examined at five freeze-thaw cycles between –65/−20◦C and room temperature. The longterm stability was tested after storage of acidulated samples at −65/–20 ◦ C for 62 days. The post-exacted stability was assessed with left QC samples in the auto-sampler for 87 h at 8 ◦ C. For urine samples, the short-term stability was measured with QC samples containing 1% acetic acid (v/v) stored for 16 h at room temperature. The urine samples without acetic acid were stored at room temperature for 2.2 h and at 2–8 ◦ C for 5.4 h. The freeze-thaw stability was examined at five freeze-thaw cycles between −20 ◦ C and room temperature. The long-term stability was determined after storage of acidulated urine samples at −20 ◦ C for 35 days. The post-exacted stability was assessed with left QC samples in the auto-sampler for 48 h at 8 ◦ C. Stability study demonstrated no significant changes in QC samples.
ceedings [5,6] and the reported studies [7,8], our PK parameters are obtained from healthy Chinese volunteers. However, the PK parameters in existing literature are obtained from patients. 3.3.2. Multiple-dose study The trough plasma concentrations from day 3–5 showed no significant difference (P > 0.05, one-way ANOVA), indicating that steady state had been reached after consecutive administration for 3 days at a 12-h interval. The PK parameters are summarized in Tab 1. Compared with the single-dose study, the main PK parameters showed no significant difference. The accumulation index (RAUC ) was (1.08 ± 0.04), proving that there was no drug accumulation tendency in healthy Chinese subjects.
3.3. Pharmacokinetic results
3.3.3. Gender effects The PK parameters after single-dose and multiple-dose administration are divided by gender in Tab 2. The Cmax and AUC0–12h were corrected with dose and weight. The main PK parameters showed no significant difference between two gender groups (P > 0.05, independent-samples t-test), indicating that PK behavior of cefbuperazone had no gender difference.
3.3.1. Single-dose study Following drip intravenous infusion, plasma levels of cefbuperazone reached maximum at 60 min and rapidly eliminated. Mean plasma concentration-time profiles after single-dose administration are presented in Fig. 3. The PK parameters are reported in Table 1. No significant difference was identified in pharmacokinetic parameters of t1/2 , Vd/F, CL/F and kel between the three dose groups. There was an excellent linear relationship between AUC0−12h and dose, as well as Cmax and dose. Compared with the conference pro-
3.3.4. Urinary excretion Mean urinary cumulative excretion amount-time profiles are shown in Fig. 4. The concentration of cefbuperazone in urine samples collected during 0–2 h was highest and then decreased rapidly. About 60.0% of the administered drug was excreted via urine as unchanged form within 12 h, indicating that the dominant elimination pathway for cefbuperazone in healthy Chinese subjects was urinary excretion. Dose-normalized cumulative excretion amount showed no significant difference between the three dose groups
Table 2 Pharmacokinetic parameters of cefbuperazone in healthy Chinese volunteers (n = 12 per dose) divided by gender after single and multiple drip intravenous infusion of cefbuperazone sodium injection. 1.0 g 0.5 g Parameters −1
Cmax (g mL ) Tmax (h) AUC0–12 (g h mL-1 ) t1/2 (h) MRT0 − 12 (h) CL/F (L/h) Vd /F (L) AUCss (g h mL−1 ) Cav (g mL−1 )
Single dose
Multiple dose
2.0 g
female
male
female
male
female
male
female
male
42.4 ± 6.6 1 109 ± 20 2.0 ± 0.1 2.7 ± 0.1 4.7 ± 0.9 13.1 ± 2.4 – –
47.1 ± 9.3 1 120 ± 23 1.9 ± 0.2 2.7 ± 0.2 4.3 ± 0.8 11.8 ± 2.1 – –
93.7 ± 14.8 1 227 ± 42 1.7 ± 0.2 2.6 ± 0.2 4.5 ± 0.9 10.8 ± 1.6 – –
79.7 ± 4.3 1 215 ± 28 2.0 ± 0.2 2.9 ± 0.2 4.6 ± 0.6 13.1 ± 1.1 – –
101 ± 16 1 243 ± 46 1.7 ± 0.1 2.5 ± 0.2 4.2 ± 0.9 10.5 ± 2.0 242.9 ± 45.6 20.2 ± 3.8
89.0 ± 3.8 1 232 ± 21 2.0 ± 0.2 2.8 ± 0.2 4.3 ± 0.4 12.0 ± 1.1 232.1 ± 21.4 19.3
174 ± 14 1 425 ± 54 1.9 ± 0.2 2.7 ± 0.2 4.7 ± 0.6 12.5 ± 1.4 – –
162 ± 11 1 418 ± 68 1.9 ± 0.2 2.7 ± 0.3 4.8 ± 0.8 13.0 ± 0.7 – –
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(P > 0.05, one-way ANOVA), proving that urinary cumulative excretion was proportional to the administered dose within the range of 0.5–2.0 g. 4. Conclusion A selective and reproducible HPLC–MS/MS method for the determination of cefbuperazone in human plasma and urine was established, validated and successfully applied to a pharmacokinetic study. The pharmacokinetic profile of cefbuperazone and accumulation in healthy Chinese subjects were studied in detail. Cefbuperazone has a dose-independent pharmacokinetic profile without drug accumulation tendency. The dominant elimination pathway was urinary excretion as unchanged form. References [1] F.Z. Jia, Clinical application of cephamycin antibiotics, Pract. Geriatr. 12 (1998) 169–170. [2] J.M.T. Hamilton-Miller, W. Brumfitt, Cephamycins: a review, prospects and some original observations, Infection 3 (1975) 183–188.
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