www.jss-journal.com
Page 1
Journal of Separation Science
Quantitative analysis and pharmacokinetics study of tigecycline in human serum using a validated sensitive liquid chromatography with tandem mass spectrometry method
Jiao Xie1, Taotao Wang1, Xue Wang2, Xiaoliang Cheng1, Haiyan Dong1, Yan Wang1, Xiaowei Zheng1, Liang Zhou3, Jianfeng Xing4, Yalin Dong1*
1
Department of Pharmacy, The First Affiliated Hospital of Medical College, Xi’an Jiaotong
University, Xi’an 710061, China. 2
Central Intensive Care Unit, The First Affiliated Hospital of Medical College, Xi’an
Jiaotong University, Xi’an 710061, China. 3
ThermoFisher Scientific (China), Building 6, No.27 XinJinqiao Road, Shanghai, 201206,
China. 4
Department of Pharmacy, College of medicine, Xi’an Jiaotong University, Xi’an 710061,
China.
Received: 12-Feb-2014; Revised: 13-Mar-2014; Accepted: 15-Mar-2014. This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/jssc.201400152. This article is protected by copyright. All rights reserved.
www.jss-journal.com
Page 2
Journal of Separation Science
*Corresponding author: Yalin Dong, Ph.D.
Department of Pharmacy, The First Affiliated Hospital of Medical College, Xi’an Jiaotong University, Xi’an 710061, China. Phone: +86-29-85323241 Fax: +86-29-85323240
Email: [email protected]; [email protected]
Keywords: intensive care medicine; LC–MS/MS; pharmacokinetics; tetracycline;tigecycline
Abbreviation: CAP, community-acquired pneumonia; CE, collision energy; cIAIs, complicated intra-abdominal infections; CLss, clearance in steady state; Cmax, a peak serum concentration; Cmin, the steady-state trough serum concentration; cSSSIs, complicated skin and skin structure infections; CXP, collision exit potential; DP, declustering potential; EP, entrance potential; fAUC/MIC, the relationship between the area under the free concentration-time curve and the minimal inhibitory concentration; FDA, Food and Drug Administration; ICU, intensive care unit; IS, internal standard; LLOQ, lower limit of quantitation; PD, pharmacodynamic; PK, pharmacokinetic; QC, quality control; SRM, selected reaction monitoring;t1/2, half-life; TCA, Trichloroacetic acid; ULOQ, upper limit of quantification;
VAP,
ventilator-associated
steady-state volume of distribution. This article is protected by copyright. All rights reserved.
bacterial
pneumonia;
Vss,
www.jss-journal.com
Page 3
Journal of Separation Science
Abstract Tigecycline, a novel intravenously administered glycylcycline antibiotic, currently plays a key role in the management of complicated multi-organism infections. However, current LC–MS/MS methods briefly describe parameters and the only reported internal standard was sometimes difficult to obtain. In our study, an updated LC–MS/MS method for the quantitative analysis of tigecycline in human serum was developed. Sample preparation involved precipitation with 20% trichloroacetic acid. Chromatographic separation of tigecycline and tetracycline (internal standard) was achieved on a Hypersil GOLD C18 column using gradient elution. The selected reaction monitoring transitions were performed at m/z 586.1→ 513.2 for tigecycline and m/z 445.1 → 410.2 for tetracycline. The assay was linear over the concentration range of 5–2000 ng/mL. The intra- and inter-day precisions at three concentration levels (10, 100 and 1600 ng/mL) were lower than 15% and their accuracies were within the range of 95.1–106.1%. The mean recovery ranged from 94.3 to 105.6% and the matrix effect from 92.1 to 97.6%. Tigecycline was stable under all tested conditions. This validated method was successfully applied to a pharmacokinetic study in critically ill patients. The data demonstrated our method allows quantification of tigecycline in serum in a quick and reliable manner for widespread application. Introduction There is a constant need to combat the increasing bacterial resistance to currently existing antimicrobial agents. Strains of potentially life-threatening bacteria such as Staphylococcus aureus, the leading cause of hospital-acquired infections, that are resistant to multiple antibiotics, are becoming progressively widespread [1, 2]. A high rate of inappropriate use of This article is protected by copyright. All rights reserved.
www.jss-journal.com
Page 4
Journal of Separation Science
antimicrobials has been observed, as one of the multitudes of factors, contributing to the evolution of more and more antimicrobial-resistant bacterial species [3, 4]. Tigecycline, a novel intravenously administered glycylcycline antibiotic, remains to be a promising one that currently plays a key role in the management of complicated multi-organism infections and is approved for the treatment of complicated intra-abdominal infections (cIAIs), complicated skin and skin structure infections (cSSSIs) [5-7] and community-acquired pneumonia (CAP) [8, 9] caused by susceptible strains of indicated pathogens in adults. This antimicrobial agent has demonstrated an expanded spectrum of in vitro activity and clinical potency against gram-positive and gram-negative aerobic and anaerobic bacteria, as well as against antibiotic-resistant strains. However, several failures of tigecycline therapy have occurred in recent years, as has been seen in ventilator-associated bacterial pneumonia (VAP) and other bacterial infections [10-13]. These failures are likely due to the development of tigecycline resistance and perhaps to inadequate dosing. In response, rational tigecycline use is proposed to prevent an escalation in antimicrobial resistance and to maximize the likelihood of a favorable clinical response, as well as to minimize the probability of exposure-related toxicity. It is now well-recognized that the intrinsic pharmacokinetic (PK) and pharmacodynamic (PD) properties of the drug must be considered in order to achieve optimal clinical and microbiological outcomes [14-16]. Prior to the establishment of tigecycline PK/PD model, a rapid and sensitive method for the analysis of tigecycline in human serum is necessarily demanded. However, only two methodological reports using HPLC [17] and ultra high performance liquid chromatography (UHPLC) [18] for the determination of tigecycline in human serum were published to date. This article is protected by copyright. All rights reserved.
www.jss-journal.com
Page 5
Journal of Separation Science
The analysis of published data revealed that HPLC was extensively used for estimation of tigecycline in biological samples, with an approximately 10 min run time and generally using minocycline as internal standard (IS). Whereas the sensitivity is improved with a mass detector, also, provides the better available reliability, repeatability and analysis time in several pharmacokinetics studies [19-24] and [tert-butyl-d9] tigecycline was the only reported IS in the LC–MS/MS method. But these limited studies utilizing LC–MS/MS for tigecycline determination in human serum/plasma were described very briefly (mobile phase, flow rate and run time were not available) without validation, making them unable to be the reference method for widespread application in future clinical studies. In our present study, a newly optimized and fully validated LC–MS/MS method was established to solve the above problems for the construction of PK/PD model applying to individual therapy. The method validation was based on the US Food and Drug Administration’s
(FDA)
guidance
for
industry,
bioanalytical
method
validation
(http://www.fda.gov/cder/guidance/4252fnl.htm) and this proposed method was applied to a pharmacokinetic study in critically ill intensive care unit (ICU) patients. Furthermore, we also gave an overview of current published tigecycline analytical methods, in order to review the previous description and to present the improvement of our method.
1. Materials and methods 1.1 Chemicals and materials All chemicals and reagents were HPLC grade or analytical grade. Tigecycline (purity 96.0%) was obtained from Toronto Research Chemicals (Toronto, Ontario, Canada) (see Fig. 1-A). This article is protected by copyright. All rights reserved.
www.jss-journal.com
Page 6
Journal of Separation Science
Tetracycline hydrochloride (purity 98.0%) (IS) was obtained from Dr. Ehrenstorfer (Augsburg, Germany) (see Fig. 1-B). Acetonitrile (HPLC grade) was purchased from Merk (Germany). Formic acid, Ammonium formate and Trichloroacetic acid (TCA) were purchased from Kemiou Chemicals (Tianjin, China). Drug-free human plasma was provided by the Blood Bank of Xi’an Jiaotong University the First Affiliated Hospital.
1.2 Instrumentation The LC–MS/MS system used consisted of TSQ Vantage triple quadrupole mass spectrometer (Thermo Fisher Scientific, San Jose, CA). Chromatographic separation of tigecycline and IS was achieved on a Hypersil GOLD C18 column (100 mm × 2.1 mm, 3 μm) with a guard column using two mobile phases, a mixture solution of 5mM Ammonium formate (containing 0.2% formic acid) (A) and Acetonitrile (B). The flow rate was 0.4 ml/min for 5.0 min. The gradient elution was delivered as follows (A:B): 0–3.0 min: 95:5–20:80; 3.0–3.5min: 20:80–20:80; 3.5–3.6min: 20:80–95:5; 3.6–5.0min: 95:5–95:5. 10 μL of sample was injected into the system by auto sampler, and the column temperature was maintained at room temperature. The mass spectrometer was operated in an ESI positive ion mode. The selected reaction monitoring (SRM) transitions were performed at m/z 586.1 → 513.2 for tigecycline (see Fig. 1-C) and m/z 445.1 → 410.2 for IS (see Fig. 1-D). Optimized values for declustering potential (DP), collision energy (CE), entrance potential (EP) and collision exit potential (CXP) were 80 V, 26 eV, 10 V, 15 V and 85 V, 17 eV, 10 V, 15 V for tigecycline and IS, respectively. Other ion source conditions were as follows: Curtain Gas was 25 psi, IonSpary This article is protected by copyright. All rights reserved.
www.jss-journal.com
Page 7
Journal of Separation Science
Voltage was 3000 V, and source temperature was 320℃.
1.3 Preparation of stock solutions, calibration standard and quality control (QC) samples Stock solutions of tigecycline (1.0 mg/mL) and IS (1.0 mg/mL) were prepared in deionized water and stored at –80℃. A series of working standard solutions of tigecycline ranging from 5 to 2000 ng/mL and an IS solution at 100 ng/mL were prepared by dilutions of their stock solutions with deionized water. Calibration standard serum samples were prepared as follows: 10 μL each working standard solution was mixed with 200 μL blank human serum to obtain the concentration of 5, 10, 50, 100, 500, 1000, 2000 ng/mL. QC solutions and QC serum samples (10, 100 and 1600 ng/mL) were prepared in the same way.
1.4 Sample preparation Serum sample was prepared with protein precipitation using 20% TCA. After addition of 10 μL IS solution (100 ng/mL), 100 μL 20% TCA was added into 200 μL serum samples in 2 mL Eppendorf tubes. After a thorough vortex mixing for 2 min, the mixture was centrifuged at 12,000 rpm for 10 min, and then 10 μL of the supernatant was injected into the LC–MS/MS system.
1.5 Method validation During the process of method validation, specificity, linearity, lower limit of quantitation (LLOQ), precision, accuracy, extraction recovery, and stability were evaluated. 2.5.1 Specificity and selectivity This article is protected by copyright. All rights reserved.
www.jss-journal.com
Page 8
Journal of Separation Science
The specificity and selectivity of the method were performed by examining the presence or absence of interference, comparing chromatograms of six lots of blank human serum samples from different sources, blank serum spiked with standard, and human serum sample after intravenously administration of tigecycline. 2.5.2 Linearity, carry-over effect and LLOQ Each calibration curve should consist of a blank sample, a zero sample and six to eight calibration concentration levels, among which blank and zero samples were only analyzed to confirm the absence of interferences. Carry-over effect should be assessed by injecting blank samples following the calibration standard at the highest concentration, and the effect should not be greater than 20% of LLOQ (defined as the lowest drug concentration on the calibration curve). Calibration curves were plotted by the peak area ratio vs. analyte concentrations using a 1/X2 weighted linear least-squares regression model. The back calculated standard concentrations should be within ± 15% of the nominal value, except for the LLOQ for which it should be within ± 20%. The LLOQ was evaluated by analyzing five replicates of spiked samples at the concentration of 5 ng/mL, and the precision and accuracy (n = 5) should be within ± 20%. 2.5.3 Accuracy and precision Intra- and inter-day precisions were assessed by assay of five replicates of QC serum samples at low, medium, and high concentrations (10, 100 and 1600 ng/mL) on the same day and on three different days. The accuracy of an analytical method describes the closeness of the determined value obtained by the method to the nominal concentration of the analyte (expressed in percentage) and the precision of the analytical method describes the closeness This article is protected by copyright. All rights reserved.
www.jss-journal.com
Page 9
Journal of Separation Science
of repeated individual measures of analyte (expressed in RSD%). The acceptance criteria should be within 15% RSD and 85–115% of nominal concentration for the precision and accuracy, respectively. 2.5.4 Matrix effect and extraction recovery Matrix effects should be investigated using the matrix factor by calculating the ratio of the peak area in the presence of matrix (measured by analyzing blank matrix spiked after extraction with analyte), to the peak area in absence of matrix (pure solution of the analyte). This determination should be done at a low and at a high level of concentration (maximum of three times the LLOQ and close to the upper limit of quantification, ULOQ). The extraction efficiency is the extraction efficiency of an analytical process, reported as a percentage of the known amount of an analyte carried through the sample extraction and processing steps of the method. The extraction recovery of tigecycline was carried out at the three QC levels (10, 100 and 1600 ng/mL) in five replicates. 2.5.5 Stability Stabilities of tigecycline in human serum were estimated by assay of three replicates of QC samples at low, medium, and high concentrations (10, 100 and 1600 ng/mL) under the following conditions: short-term stability after storage at room temperature (25℃) for 4 h; long-term storage stability after storage at –80℃ for 15 and 30 days; freeze–thaw stability through three freeze–thaw cycles (–80 to 25℃). The post-preparative stability was examined after 24 h in the autosampler maintained at 25℃. The stock solution stabilities of tigecycline and IS were demonstrated following storage at –80℃. The measured concentrations of stabilities are compared to the nominal concentrations. The relative standard deviation should This article is protected by copyright. All rights reserved.
www.jss-journal.com
Page 10
Journal of Separation Science
be within ± 15%.
1.6 Application to pharmacokinetic study The steady-state pharmacokinetic study was approved by Xian Jiaotong University the First Affiliated Hospital. All subjects signed the informed consent before any screening item being performed. In this clinical study, 54 blood samples collected from a total of six critically ill patients were measured, including five men and one woman with average age of 58 (range, 35–75). All of these patients were suffered from pulmonary infections caused by multidrug-resistant
Acinetobacter
baumannii
and
the initial
i.v. dose of 100 mg
tigecycline was administered followed by 50 mg q12h over 30 min for at least five days. Steady-state drug levels were attained before the seventh dose and the steady-state concentrations of blood collection points are as follows: 0 (pre-dose), 0.5, 1, 2, 3, 4, 6, 8 and 12 h. Volumes of 1 mL of blood samples were collected in Vacutainer tubes (BD, Franklin Lakes, NJ) and were immediately centrifuged at 3500 rpm for 5 min and the supernatants were stored frozen at –80℃ until analysis. Steady-state pharmacokinetic parameters were calculated by non-compartmental model using Winnonlin software (version 4.1, Pharsight Corporation).
2. Results and discussion 3.1 Specificity and selectivity The representative chromatograms of a human blank serum spiked with tigecycline (LLOQ, 5 ng/mL) and IS, and a serum sample collected at 8 h after multi-doses intravenously This article is protected by copyright. All rights reserved.
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Journal of Separation Science
administration of 50 mg q12h tigecycline to patients were shown in Fig. 2. No interference peaks from endogenous substances were observed at the retention times of tigecycline and IS in the chromatograms, indicating good specificity and selectivity of the method.
3.2 Linearity, carry-over effect and LLOQ The calibration curve for tigecycline in human serum was linear over the concentration range of 5–2000 ng/mL. The linear regression equation (n = 7) was Y = 0.0394X – 0.1356, with correlation coefficients (r2= 0.9978) of the regression equation greater than 0.99 in all cases. No peaks in the chromatographic region of the analytes of interest were observed by injecting blank plasma extract immediately after ULOQ sample, indicating that carry-over effect from previous concentrated samples was negligible. The LLOQ for tigecycline was 5 ng/mL and the accuracy (%) and precision (RSD, %) was 103.5 and 1.1%, respectively.
3.3 Accuracy and precision Under the current method validation conditions, a satisfactory precision, accuracy and reproducibility of tigecycline in human serum were obtained by analyzing QC samples at low, medium and high levels. The inter- and inter-day precisions (RSD, %) were
Page 1
Journal of Separation Science
Quantitative analysis and pharmacokinetics study of tigecycline in human serum using a validated sensitive liquid chromatography with tandem mass spectrometry method
Jiao Xie1, Taotao Wang1, Xue Wang2, Xiaoliang Cheng1, Haiyan Dong1, Yan Wang1, Xiaowei Zheng1, Liang Zhou3, Jianfeng Xing4, Yalin Dong1*
1
Department of Pharmacy, The First Affiliated Hospital of Medical College, Xi’an Jiaotong
University, Xi’an 710061, China. 2
Central Intensive Care Unit, The First Affiliated Hospital of Medical College, Xi’an
Jiaotong University, Xi’an 710061, China. 3
ThermoFisher Scientific (China), Building 6, No.27 XinJinqiao Road, Shanghai, 201206,
China. 4
Department of Pharmacy, College of medicine, Xi’an Jiaotong University, Xi’an 710061,
China.
Received: 12-Feb-2014; Revised: 13-Mar-2014; Accepted: 15-Mar-2014. This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/jssc.201400152. This article is protected by copyright. All rights reserved.
www.jss-journal.com
Page 2
Journal of Separation Science
*Corresponding author: Yalin Dong, Ph.D.
Department of Pharmacy, The First Affiliated Hospital of Medical College, Xi’an Jiaotong University, Xi’an 710061, China. Phone: +86-29-85323241 Fax: +86-29-85323240
Email: [email protected]; [email protected]
Keywords: intensive care medicine; LC–MS/MS; pharmacokinetics; tetracycline;tigecycline
Abbreviation: CAP, community-acquired pneumonia; CE, collision energy; cIAIs, complicated intra-abdominal infections; CLss, clearance in steady state; Cmax, a peak serum concentration; Cmin, the steady-state trough serum concentration; cSSSIs, complicated skin and skin structure infections; CXP, collision exit potential; DP, declustering potential; EP, entrance potential; fAUC/MIC, the relationship between the area under the free concentration-time curve and the minimal inhibitory concentration; FDA, Food and Drug Administration; ICU, intensive care unit; IS, internal standard; LLOQ, lower limit of quantitation; PD, pharmacodynamic; PK, pharmacokinetic; QC, quality control; SRM, selected reaction monitoring;t1/2, half-life; TCA, Trichloroacetic acid; ULOQ, upper limit of quantification;
VAP,
ventilator-associated
steady-state volume of distribution. This article is protected by copyright. All rights reserved.
bacterial
pneumonia;
Vss,
www.jss-journal.com
Page 3
Journal of Separation Science
Abstract Tigecycline, a novel intravenously administered glycylcycline antibiotic, currently plays a key role in the management of complicated multi-organism infections. However, current LC–MS/MS methods briefly describe parameters and the only reported internal standard was sometimes difficult to obtain. In our study, an updated LC–MS/MS method for the quantitative analysis of tigecycline in human serum was developed. Sample preparation involved precipitation with 20% trichloroacetic acid. Chromatographic separation of tigecycline and tetracycline (internal standard) was achieved on a Hypersil GOLD C18 column using gradient elution. The selected reaction monitoring transitions were performed at m/z 586.1→ 513.2 for tigecycline and m/z 445.1 → 410.2 for tetracycline. The assay was linear over the concentration range of 5–2000 ng/mL. The intra- and inter-day precisions at three concentration levels (10, 100 and 1600 ng/mL) were lower than 15% and their accuracies were within the range of 95.1–106.1%. The mean recovery ranged from 94.3 to 105.6% and the matrix effect from 92.1 to 97.6%. Tigecycline was stable under all tested conditions. This validated method was successfully applied to a pharmacokinetic study in critically ill patients. The data demonstrated our method allows quantification of tigecycline in serum in a quick and reliable manner for widespread application. Introduction There is a constant need to combat the increasing bacterial resistance to currently existing antimicrobial agents. Strains of potentially life-threatening bacteria such as Staphylococcus aureus, the leading cause of hospital-acquired infections, that are resistant to multiple antibiotics, are becoming progressively widespread [1, 2]. A high rate of inappropriate use of This article is protected by copyright. All rights reserved.
www.jss-journal.com
Page 4
Journal of Separation Science
antimicrobials has been observed, as one of the multitudes of factors, contributing to the evolution of more and more antimicrobial-resistant bacterial species [3, 4]. Tigecycline, a novel intravenously administered glycylcycline antibiotic, remains to be a promising one that currently plays a key role in the management of complicated multi-organism infections and is approved for the treatment of complicated intra-abdominal infections (cIAIs), complicated skin and skin structure infections (cSSSIs) [5-7] and community-acquired pneumonia (CAP) [8, 9] caused by susceptible strains of indicated pathogens in adults. This antimicrobial agent has demonstrated an expanded spectrum of in vitro activity and clinical potency against gram-positive and gram-negative aerobic and anaerobic bacteria, as well as against antibiotic-resistant strains. However, several failures of tigecycline therapy have occurred in recent years, as has been seen in ventilator-associated bacterial pneumonia (VAP) and other bacterial infections [10-13]. These failures are likely due to the development of tigecycline resistance and perhaps to inadequate dosing. In response, rational tigecycline use is proposed to prevent an escalation in antimicrobial resistance and to maximize the likelihood of a favorable clinical response, as well as to minimize the probability of exposure-related toxicity. It is now well-recognized that the intrinsic pharmacokinetic (PK) and pharmacodynamic (PD) properties of the drug must be considered in order to achieve optimal clinical and microbiological outcomes [14-16]. Prior to the establishment of tigecycline PK/PD model, a rapid and sensitive method for the analysis of tigecycline in human serum is necessarily demanded. However, only two methodological reports using HPLC [17] and ultra high performance liquid chromatography (UHPLC) [18] for the determination of tigecycline in human serum were published to date. This article is protected by copyright. All rights reserved.
www.jss-journal.com
Page 5
Journal of Separation Science
The analysis of published data revealed that HPLC was extensively used for estimation of tigecycline in biological samples, with an approximately 10 min run time and generally using minocycline as internal standard (IS). Whereas the sensitivity is improved with a mass detector, also, provides the better available reliability, repeatability and analysis time in several pharmacokinetics studies [19-24] and [tert-butyl-d9] tigecycline was the only reported IS in the LC–MS/MS method. But these limited studies utilizing LC–MS/MS for tigecycline determination in human serum/plasma were described very briefly (mobile phase, flow rate and run time were not available) without validation, making them unable to be the reference method for widespread application in future clinical studies. In our present study, a newly optimized and fully validated LC–MS/MS method was established to solve the above problems for the construction of PK/PD model applying to individual therapy. The method validation was based on the US Food and Drug Administration’s
(FDA)
guidance
for
industry,
bioanalytical
method
validation
(http://www.fda.gov/cder/guidance/4252fnl.htm) and this proposed method was applied to a pharmacokinetic study in critically ill intensive care unit (ICU) patients. Furthermore, we also gave an overview of current published tigecycline analytical methods, in order to review the previous description and to present the improvement of our method.
1. Materials and methods 1.1 Chemicals and materials All chemicals and reagents were HPLC grade or analytical grade. Tigecycline (purity 96.0%) was obtained from Toronto Research Chemicals (Toronto, Ontario, Canada) (see Fig. 1-A). This article is protected by copyright. All rights reserved.
www.jss-journal.com
Page 6
Journal of Separation Science
Tetracycline hydrochloride (purity 98.0%) (IS) was obtained from Dr. Ehrenstorfer (Augsburg, Germany) (see Fig. 1-B). Acetonitrile (HPLC grade) was purchased from Merk (Germany). Formic acid, Ammonium formate and Trichloroacetic acid (TCA) were purchased from Kemiou Chemicals (Tianjin, China). Drug-free human plasma was provided by the Blood Bank of Xi’an Jiaotong University the First Affiliated Hospital.
1.2 Instrumentation The LC–MS/MS system used consisted of TSQ Vantage triple quadrupole mass spectrometer (Thermo Fisher Scientific, San Jose, CA). Chromatographic separation of tigecycline and IS was achieved on a Hypersil GOLD C18 column (100 mm × 2.1 mm, 3 μm) with a guard column using two mobile phases, a mixture solution of 5mM Ammonium formate (containing 0.2% formic acid) (A) and Acetonitrile (B). The flow rate was 0.4 ml/min for 5.0 min. The gradient elution was delivered as follows (A:B): 0–3.0 min: 95:5–20:80; 3.0–3.5min: 20:80–20:80; 3.5–3.6min: 20:80–95:5; 3.6–5.0min: 95:5–95:5. 10 μL of sample was injected into the system by auto sampler, and the column temperature was maintained at room temperature. The mass spectrometer was operated in an ESI positive ion mode. The selected reaction monitoring (SRM) transitions were performed at m/z 586.1 → 513.2 for tigecycline (see Fig. 1-C) and m/z 445.1 → 410.2 for IS (see Fig. 1-D). Optimized values for declustering potential (DP), collision energy (CE), entrance potential (EP) and collision exit potential (CXP) were 80 V, 26 eV, 10 V, 15 V and 85 V, 17 eV, 10 V, 15 V for tigecycline and IS, respectively. Other ion source conditions were as follows: Curtain Gas was 25 psi, IonSpary This article is protected by copyright. All rights reserved.
www.jss-journal.com
Page 7
Journal of Separation Science
Voltage was 3000 V, and source temperature was 320℃.
1.3 Preparation of stock solutions, calibration standard and quality control (QC) samples Stock solutions of tigecycline (1.0 mg/mL) and IS (1.0 mg/mL) were prepared in deionized water and stored at –80℃. A series of working standard solutions of tigecycline ranging from 5 to 2000 ng/mL and an IS solution at 100 ng/mL were prepared by dilutions of their stock solutions with deionized water. Calibration standard serum samples were prepared as follows: 10 μL each working standard solution was mixed with 200 μL blank human serum to obtain the concentration of 5, 10, 50, 100, 500, 1000, 2000 ng/mL. QC solutions and QC serum samples (10, 100 and 1600 ng/mL) were prepared in the same way.
1.4 Sample preparation Serum sample was prepared with protein precipitation using 20% TCA. After addition of 10 μL IS solution (100 ng/mL), 100 μL 20% TCA was added into 200 μL serum samples in 2 mL Eppendorf tubes. After a thorough vortex mixing for 2 min, the mixture was centrifuged at 12,000 rpm for 10 min, and then 10 μL of the supernatant was injected into the LC–MS/MS system.
1.5 Method validation During the process of method validation, specificity, linearity, lower limit of quantitation (LLOQ), precision, accuracy, extraction recovery, and stability were evaluated. 2.5.1 Specificity and selectivity This article is protected by copyright. All rights reserved.
www.jss-journal.com
Page 8
Journal of Separation Science
The specificity and selectivity of the method were performed by examining the presence or absence of interference, comparing chromatograms of six lots of blank human serum samples from different sources, blank serum spiked with standard, and human serum sample after intravenously administration of tigecycline. 2.5.2 Linearity, carry-over effect and LLOQ Each calibration curve should consist of a blank sample, a zero sample and six to eight calibration concentration levels, among which blank and zero samples were only analyzed to confirm the absence of interferences. Carry-over effect should be assessed by injecting blank samples following the calibration standard at the highest concentration, and the effect should not be greater than 20% of LLOQ (defined as the lowest drug concentration on the calibration curve). Calibration curves were plotted by the peak area ratio vs. analyte concentrations using a 1/X2 weighted linear least-squares regression model. The back calculated standard concentrations should be within ± 15% of the nominal value, except for the LLOQ for which it should be within ± 20%. The LLOQ was evaluated by analyzing five replicates of spiked samples at the concentration of 5 ng/mL, and the precision and accuracy (n = 5) should be within ± 20%. 2.5.3 Accuracy and precision Intra- and inter-day precisions were assessed by assay of five replicates of QC serum samples at low, medium, and high concentrations (10, 100 and 1600 ng/mL) on the same day and on three different days. The accuracy of an analytical method describes the closeness of the determined value obtained by the method to the nominal concentration of the analyte (expressed in percentage) and the precision of the analytical method describes the closeness This article is protected by copyright. All rights reserved.
www.jss-journal.com
Page 9
Journal of Separation Science
of repeated individual measures of analyte (expressed in RSD%). The acceptance criteria should be within 15% RSD and 85–115% of nominal concentration for the precision and accuracy, respectively. 2.5.4 Matrix effect and extraction recovery Matrix effects should be investigated using the matrix factor by calculating the ratio of the peak area in the presence of matrix (measured by analyzing blank matrix spiked after extraction with analyte), to the peak area in absence of matrix (pure solution of the analyte). This determination should be done at a low and at a high level of concentration (maximum of three times the LLOQ and close to the upper limit of quantification, ULOQ). The extraction efficiency is the extraction efficiency of an analytical process, reported as a percentage of the known amount of an analyte carried through the sample extraction and processing steps of the method. The extraction recovery of tigecycline was carried out at the three QC levels (10, 100 and 1600 ng/mL) in five replicates. 2.5.5 Stability Stabilities of tigecycline in human serum were estimated by assay of three replicates of QC samples at low, medium, and high concentrations (10, 100 and 1600 ng/mL) under the following conditions: short-term stability after storage at room temperature (25℃) for 4 h; long-term storage stability after storage at –80℃ for 15 and 30 days; freeze–thaw stability through three freeze–thaw cycles (–80 to 25℃). The post-preparative stability was examined after 24 h in the autosampler maintained at 25℃. The stock solution stabilities of tigecycline and IS were demonstrated following storage at –80℃. The measured concentrations of stabilities are compared to the nominal concentrations. The relative standard deviation should This article is protected by copyright. All rights reserved.
www.jss-journal.com
Page 10
Journal of Separation Science
be within ± 15%.
1.6 Application to pharmacokinetic study The steady-state pharmacokinetic study was approved by Xian Jiaotong University the First Affiliated Hospital. All subjects signed the informed consent before any screening item being performed. In this clinical study, 54 blood samples collected from a total of six critically ill patients were measured, including five men and one woman with average age of 58 (range, 35–75). All of these patients were suffered from pulmonary infections caused by multidrug-resistant
Acinetobacter
baumannii
and
the initial
i.v. dose of 100 mg
tigecycline was administered followed by 50 mg q12h over 30 min for at least five days. Steady-state drug levels were attained before the seventh dose and the steady-state concentrations of blood collection points are as follows: 0 (pre-dose), 0.5, 1, 2, 3, 4, 6, 8 and 12 h. Volumes of 1 mL of blood samples were collected in Vacutainer tubes (BD, Franklin Lakes, NJ) and were immediately centrifuged at 3500 rpm for 5 min and the supernatants were stored frozen at –80℃ until analysis. Steady-state pharmacokinetic parameters were calculated by non-compartmental model using Winnonlin software (version 4.1, Pharsight Corporation).
2. Results and discussion 3.1 Specificity and selectivity The representative chromatograms of a human blank serum spiked with tigecycline (LLOQ, 5 ng/mL) and IS, and a serum sample collected at 8 h after multi-doses intravenously This article is protected by copyright. All rights reserved.
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administration of 50 mg q12h tigecycline to patients were shown in Fig. 2. No interference peaks from endogenous substances were observed at the retention times of tigecycline and IS in the chromatograms, indicating good specificity and selectivity of the method.
3.2 Linearity, carry-over effect and LLOQ The calibration curve for tigecycline in human serum was linear over the concentration range of 5–2000 ng/mL. The linear regression equation (n = 7) was Y = 0.0394X – 0.1356, with correlation coefficients (r2= 0.9978) of the regression equation greater than 0.99 in all cases. No peaks in the chromatographic region of the analytes of interest were observed by injecting blank plasma extract immediately after ULOQ sample, indicating that carry-over effect from previous concentrated samples was negligible. The LLOQ for tigecycline was 5 ng/mL and the accuracy (%) and precision (RSD, %) was 103.5 and 1.1%, respectively.
3.3 Accuracy and precision Under the current method validation conditions, a satisfactory precision, accuracy and reproducibility of tigecycline in human serum were obtained by analyzing QC samples at low, medium and high levels. The inter- and inter-day precisions (RSD, %) were
