Development and validation of a sensitive LC/MS-MS method for the determination of letrozole in nude mice plasma and its application to a pharmacokinetic study 
Junsheng Xue2, Qingyu Yao2, Jian Li2, Wenjun Chen2, Hong Su2, Xiuyun Tian3, Chunyi Hao3, Tianyan Zhou1,2*          
1. Beijing Key Laboratory of molecular Pharmaceutics and New Drug Delivery Systems, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100191, China
2. Department of Pharmaceutics, School of Pharmaceutical Science, Peking University Health Science Center, Beijing 100191, China
3. Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing), Department of Hepato-Pancreato-Biliary Surgery, Peking University Cancer Hospital & Institute, Beijing 100142, China
Abstract: A sensitive, rapid and simple liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed and validated for the determination of letrozole (LTZ) in nude mouse plasma in the current study, which was successfully applied to a pharmacokinetic study. Using anastrozole as internal standard (IS), plasma samples went through a one-step protein precipitation with acetonitrile before determination. The analyte and IS were analyzed on a reversed-phase ZORBAX-SB-C18column (4.6 mm×250 mm, 5 μm) with an isocratic mobile phase consisting of acetonitrile and water containing 0.1% formic acid (v/v) at a flow rate of 1.0 mL/min. The analyte and IS were detected by a triple-quadrupole tandem mass spectrometer, and electrospray and multiple reaction monitoring (MRM) were employed to select LTZ at m/z 286.4/217.1 and IS at m/z 294.1/225.3 simultaneously in the positive ion mode. The calibration curve showed good linearity ranging from 0.8–2000.0 ng/mL (r>0.99). The intra-day and inter-day precisions of LTZ were 4.0%–8.4%, with an accuracy of 98.6%–104.9%. Using this method, we successfully characterized the pharmacokinetics (PK) of LTZ by a one-compartment model with first-order absorption in female BALB/c nude mice. 
Keywords: LC-MS/MS; Letrozole; Nude mice; Pharmacokinetics   
CLC number: R943                Document code: A                 Article ID: 10031057(2018)1066510
1. Introduction
Estrogen plays a vital role in various physiological processes, such as maintaining female secondary sex characteristics[1], meditating the maturity of bone system and cardiovascular system[2,3]. Aromatase is an essential enzyme responsible for the conversion of testosterone into 17β-estradiol[4], the most active estrogens in the body[5]. Abundant evidence shows that the aromatase expression is significantly increased in hormone-sensitive breast cancer, leading to a high concentration of estrogen in carcinoma tissue, which ultimately promotes tumor growth[6–9]. Therefore, the inhibition of aromatase can be a strategy for breast cancer treatment. Letrozole (LTZ) (Fig. 1A), a third-generation non-steroidal aromatase inhibitor with high potency, can competitively and reversibly bind to aromatase to block the synthesis of estrogen[10]. Several preclinical studies have proved the excellent anti-tumor effect of LTZ in breast cancer xenografts[11–13]. Due to its superioranti-tumor efficacy and safety, LTZ has been widely used in the adjuvant therapy of estrogen receptor positive breast cancer in post-menopausal women[14,15].
Figure 1. Chemical structures of LTZ (A) and anastrozole (IS, B). 
The investigation of PK is an indispensable procedure during drug development. Based on the PK properties of a certain drug, pharmacokinetic/pharmacodynamic (PK/PD) modeling can be applied to describe the relationship between drug exposure and efficacy quantitatively, which does great help to dose regimen optimization and extrapolation from animals to human[16–19]. Several PK studies of LTZ have been reported in human[20–22]and rats[23–25]. However, the PK behavior in nude mice is still unknown. As we known, nude mice are one of the most standard animals used in the preclinical research of anti-tumor drugs. Meanwhile, PK properties of LTZ vary from human to rats, and even the gender can be a factor resulting in the difference of PK behavior[23,26]. Therefore, it is of great necessity to investigate LTZ’s PK in nude mice.
The existing analytical methods for LTZ are mainly for human or rat plasma[23,27,28], but lacking in nude mice, which hinders the in vivo preclinical study of LTZ, especially PK/PD study. In addition, the reported methods using high-performance liquid chromatographic (HPLC) show a relatively high lower limit of quantitation (LLOQ) of 3150 ng/mL[28–30]. Besides, several methods using LC-MS/MS methods are based on multi-step sample preparation procedures[26,28,31],which are time-consuming.
Here in this study, we aimed to develop and validate a sensitive and rapid LC-MS/MS method with one-step protein precipitation sample preparation procedure for the determination of LTZ in nude mouse plasma.
2. Materials and methods
2.1. Reagents and chemicals
LTZ (>98%) and anastrozole (>98%, Fig. 1B) were purchased from Energy Chemical (Shanghai, China). Methanol and acetonitrile (HPLC-grade) were obtained from Merck Co., Inc. (Darmstadt, Germany). Formic acid (>98%) was provided by Beijing Chemical Works (Beijing, China). Hydroxypropyl-β-cyclodextrin (HP-β-CD) was purchased from Dalian Meilun Biotech Co., Ltd. (Dalian, China). The distilled water used in this study was obtained from Watson’s Food & Drinks Co., Ltd. (Guangzhou, China). Cell-grade dimethyl sulfoxide (DMSO) was supplied by PanReac AppliChem Co., Inc. (Gatersleben, Germany). Blank nude mouse plasma was manually collected from female BALB/c nude mice (details in 2.6).
2.2. Instruments
The LC-MS/MS equipment consisting of a Dionex UltiMate® 3000 Ultra-HPLC system (Thermo Fisher Scientific Inc., Sunnyvale, CA, USA) and an API 4000 QTRAP mass spectrometer (Applied Biosystems Inc., Foster City, CA, USA), equipped with an electrospray ionization (ESI) source system was used in this study. The system control and data analysis were performed with Chromeleon™ Chromatography Data System (CDS) software (Thermo Fisher Scientific Inc.) and Analyst®software (Applied Biosystems Inc., Version 1.6), respectively. LTZ and IS were analyzed on a reversed-phase ZORBAX-SB-C18 Column (4.6 mm×250 mm, 5 μm, Agilent Inc., USA), protected by a Prevail™ C18 guard column (7.5 mm×2.1 mm, 3 μm, USA). Experimental data were further conducted and analyzed by NONMEM 7.3.0 (ICON Development Solutions, USA), Microsoft Excel 2016 (Microsoft Inc., Redmond, CA, USA) and GraphPad Prism (GraphPad Software, Inc., La Jolla, CA, USA, Version 5.01).  
2.3. LC-MS/MS conditions
2.3.1. HPLC conditions
The mobile phase consisted of acetonitrile (60%, v/v) and water containing 0.1% formic acid (40%, v/v). Theflow rate was set at 1.0 mL/min. Samples were kept in the dark in the autosampler at room temperature until determination. The total run time was 5.0 min with a 5-μL injection volume for each single run.
2.3.2. MS/MS conditions
LTZ and IS were monitored by a mass spectrometer with ESI in the positive ion mode and MRM mode. LTZ was monitored at m/z 286.4/217.1 with a declustering potential (DP) of 40 V and a collision energy (CE) of 16 V. IS was monitored at m/z 294.1/225.3 with a DP of 73 V and a CE of 30 V. The optimized working parameters were as follows: ion spray voltage, 5500 V; temperature, 500 °C; ion source gas 1, 60 psi; ion source gas 2, 50 psi; curtain gas, 15 psi; collision gas, 2 psi.
2.4. Solution preparation and sample pretreatment
2.4.1. Preparation of stock and working solutions
The stock solutions of LTZ and IS were prepared by dissolving in methanol to meet the concentration of 0.2 mg/mL, respectively. The stock solution of LTZ was serially diluted with methanol to generate working solutions at the concentrations of 40, 32, 8, 1.6, 0.4, 0.08, 0.04 and 0.016 μg/mL. According to flow injection analysis (FIA) tuning optimization results, IS workingsolution was further diluted to the concentration of 4 μg/mL with methanol. The stock solutions and workingsolutions were stored at 4 °C in the dark before use.
2.4.2. Preparation of calibrators and quality control samples
Using 1.5-mL Eppendorf tubes, 5 μL LTZ working solution and 5 μL IS (4 μg/mL) were added to95 μL blank nude mouse plasma to meet the finalconcentrations of 0.8, 2, 4, 20, 80, 400, 1600 and 2000 ng/mL. The mixture was then vortex-mixed for 1 min, followed by a one-step protein precipitation with 300 μL acetonitrile. After another 1 min for vortex-mixing, the mixture was centrifuged at 4 °C 14 000 r/min for 15 min. A volume of 150 μL clear supernatant was pipetted into a 250-μL polypropylene insert placed in a glass auto-injector vial. Finally, 5 μL of the clear supernatant was injected for LC-MS/MS analysis. Three concentrations of quality control samples (QCs, QC-low, -medium, and -high: 2, 400 and 1600 ng/mL, respectively) were prepared using the method as mentioned above.
2.4.3. Preparation of PK samples
PK plasma samples were stored at –80 °C and thawed at room temperature before using. Briefly, 5 μL IS was added to100 μL plasma sample. The following procedures were described in 2.4.2.
2.5. Method validation
According to guidance for bioanalytical method validation published by FDA in 2013, QC samples were processed in six replicates at each concentration to validate the selectivity, linearity, precision, accuracy, matrix effects, recovery and stability. The relative standard deviation (RSD) of each QC level should not exceed 15%.
2.5.1. Specificity
The test for specificity of this method was evaluated by six individual blank nude mouse plasma samples. These samples included blank plasma, blank plasma spiked with LTZ and IS, and plasma samples collected after administration. There should not be any interfering peaks of endogenous substances, decomposition products, metabolites or concomitant medication at the retention time of the analyte and IS, according to FDA guideline.
2.5.2. Linearity and LLOQ
The linearity of this method was validated by three calibration curves with a linear regression of 1/x by the area ratio responses for LTZ and IS on three separate days. The LLOQ was evaluated by six samples at the concentration of 0.8 ng/mL, the lowest calibration standard. The accuracy (80%–120%) and precision (≤20%) of LLOQ were acceptable according to the FDA guideline.
2.5.3. Precision and accuracy
To access the intra- and inter-day precision and accuracy, six replicate samples of QCs at three concentrations(2, 400, 1600 ng/mL) were determined in three runs in the same day and three separate days, respectively. RSDs were calculated for precision, while relative errors (REs) represented accuracy. For QC of each concentration, a precision within 15% and an accuracy within 85%–115% were acceptable.
2.5.4. Matrix effect and recovery
Blank nude mouse plasma was precipitated with acetonitrile as described previously. After centrifugation, the clear supernatant was collected. The supernatant was used for replacing blank nude plasma to prepare the QCs of three concentrations, which was added with LTZ working solution and IS. The matrix effect was calculated by the ratio of peak area responses of LTZ obtained above and working solutions of LTZ at the corresponding concentrations. The IS-normalized matrixeffect should be within 15%. The recovery was assessed by making a comparison of the peak area of processed samples with blank nude mouse plasma extracts spiked with LTZ solution at three QC concentrations. Six samples for each QC concentration were conducted.
2.5.5. Stability
The stability of LTZ in nude mouse plasma was evaluated under three storage and process conditions at three QC concentrations. For room temperature stability, six replicates were prepared and kept at room temperature for 12 h. Freeze-thaw stability was assessed by three freeze-thaw cycles (–80 °C to room temperature). For long-term stability, the QC samples were stored at –80 °C for 4 weeks before determination. The analyte was considered to be stable when RSDs were less than 15% and REs were between 85%–115%.
2.6. PK study
The validated method was applied to determine the concentration of LTZ in plasma obtained from a single-dose of 2 mg/kg PK study in female BALB/c nude mice. Briefly,16–20 g BALB/c nude mice were purchased from Animal Service of Health Science Center, Peking University and fed in individually ventilated cages (IVCs) with constant temperature (25–28 °C) and humidity (50%–60%) on a controlled 12-h light/dark cycle. All the nude mice had free access to water and rodent chow. The animals were forbidden to eat for 12 h before administration, during which only water was available. LTZ was firstly dissolved in DMSO, followed by dilution with 10% HP-β-CD, and the DMSO concentration was maintained less than 1% (v/v). After intragastric administration (i.g.), blood samples were collected into heparinized tubes at the time point of 0 (blank plasma), 0.25, 1, 1.5, 2, 4, 8, 16, 24 and 48 h. The blood samples then went through centrifugation immediately at 4000 r/min for 10 min at room temperature. Next, the clear supernatant was pipetted into 1.5-mL polypropylene tubes and stored at –80 °C until analysis. NONMEM 7.3.0 was used to calculate PK profiles of LTZ in female BALB/c nude mice. All the animal studies were approved by animal ethics of the Institutional Animal Care and Use Committee of Peking University.
3. Results and discussion
3.1. Method development
Anastrozole was selected to be IS due to its similarity in chemical structure and chromatographic profiles with LTZ based on a previous report[27]. Using Quantitative Optimization wizard in Analyst® software, the MS/MS conditions were optimized to achieve maximum responses of LTZ and IS with reasonable parameters. The finaloptimized precursor-to-product ion transitions monitoredfor LTZ and IS were m/z 286.4/217.1 and m/z 294.1/225.3, respectively, which were also similar with existing reports[27,32].
As for HPLC condition optimization, different mobile phase systems were tried on a reversed-phase ZORBAX-SB-C18 Column. The mobile phase system consisting of water and acetonitrile (40:60, v/v) showed relatively appropriate retention time and peak shape. Formic acid was employed to help with ionization of LTZ and IS and prevent the peak from tailing.
One-step protein precipitation with acetonitrile was used for sample pretreatment, which is a frequently-usedmethod in studies published by our group[33–36]. Compared with solid-phase extractions adopted by some LC-MS/MS methods[26,28,31], protein precipitation is much simpler and less time-consuming, providing acceptable matrix effects in detecting LTZ.
Since the existing methods for LTZ determination in plasma have relatively high LLOQ or complicated sample pretreatment procedures, and there are few methodsfor nude mouse plasma, as mentioned before. The improvement of sensitivity and sample pretreatment allowed the wide application to PK study.
3.2. Method validation
3.2.1. Specificity
Figure 2 illustrates the chromatograms of blank plasma, blank plasma spiked with LTZ and IS, and plasma samples collected after administration under typical MRM mode. The retention times of LTZ and IS were 3.52 min and 3.45 min, respectively. No significant endogenous interference was found at the retention time of LTZ and IS, but we failed to identify the substance corresponding to the irregular peak at the retention time of about 2.25 min. The specificity of this method was considered acceptable.
3.2.2. Linearity and LLOQ
Excellent linearity was observed over the concentration range of 0.8–2000 ng/mL of LTZ in nude mouse plasma. The calibration model could be chosen based on data analysis by linear regression with or without intercepts and weighting factors (1/x, 1/x2 and logx). For the current method, a 1/x weighting factor was finally selected to achieve linear regression. The typical linear equation was as follows: y = 0.00123x + 0.000838 (r = 0.9999), where y represents the peak area ratio of LTZ to IS, and x represents the concentration of LTZ.
The LLOQ of the established method was 0.8 ng/mL. The accuracy (RE) and the precision (RSD) were 97.2%and 13.9%, respectively, which was sensitive and accurate enough for the quantification of LTZ in nude mouse plasma according to FDA guidance.
3.2.3. Precision and accuracy
As shown in Table 1, the intra- and inter-day precisions ranged from 4.0% to 8.4%, while the accuracy fell in the range of 98.6%–104.9%. All the RSD and RE values met the requirement of FDA guideline, indicating an acceptable precision and accuracy of this method.
3.2.4. Matrix effect and recovery
Table 2 shows the summary results of matrix effect and recovery. The IS-normalized matrix effect ranged from 91.4% to 99.7%. No significant matrix effect was observed in the current method. The recovery ranged from 88.1% to 94.9%. Therefore, recovery of the method was proved to be efficient.
3.2.5. Stability
The stability results of this method were displayed in Table 3. LTZ was found to be stable at room temperature for 12 h, at –80 °C for 4 weeks and after three freeze-thaw cycles at –80 °C and room temperature with RSDs and accuracy of the determined concentrations within 0.7%–6.5% and 87.5%–99.1%.  
Figure 2. Multiple reaction monitoring chromatograms of (A) blank plasma; (B) blank plasma spiked with 8 μg/mL of LTZ and 4 μg/mL of IS; and (C) plasma sample of female nude mice collected at 4 h after administion. 


Table 1. Intra- and Inter-day precision and accuracy of LTZ (n = 6).


Table 2. Matrix effect and recovery of LTZ (n = 6).


Table 3. Stability of LTZ in plasma (n = 6).


3.3. Application in PK study
The developed and validated method was successfully applied to a preclinical PK study in female BALB/c nude mice. Figure 3 shows the plasma concentration-time profile of LTZ after a single i.g. dose of 2 mg/kg. LTZ concentrations of all samples were within the calibration curve, and no samples were diluted with blank nudemouse plasma before protein precipitation. Hence, the dilution integrity was not included in method validation. 
Figure 3. The concentration-time profile of LTZ in female BALB/c nude mice plasma after a single dose of i.g. administration of 2 mg/kg LTZ (n = 3, mean±SD).  
One compartment model with first-order absorption was employed to characterize the PK of LTZ in female BALB/c nude mice. Table 4 shows the PK model parameters estimated by NONMEM. A two-compartmentmodel structure with mixed first and zero order absorption and first order elimination has been used to characterize the concentration-time profile in a populationPK study of LTZ in healthy volunteers[37], which is different from our results. Meanwhile, great differences in PK parameters have been observed between human, rats and nude mice. For instance, the estimated clearance in nude mice was 0.139 L/kg/h, smaller than that in rat (0.185 L/kg/h) and human (1.50 L/kg/h). The terminal phase half-life was calculated as 6.68 h and Tmax was observed as 2 h in nude mice, which were much shorter than those in human andrats, indicating faster absorption and elimination processes[23,26,37]. Owing to the remarkable differences in PK profiles, it is of great necessity to investigate the PK of LTZ in nude mice.


Table 4. PK estimates of LTZ in nude mice at a i.g. dose of 2 mg/kg (n = 3).


4. Conclusions
In this study, we established and validated a sensitiveand simple LC-MS/MS method for LTZ determination in female nude mouse plasma, which was successfullyapplied to a PK study. This method had a wide quantitativerange and met the criteria for accuracy, precision, matrix effect, extraction recovery and stability as required by FDA guideline. One-compartment model with first-orderabsorption best characterized the PK of LTZ. This work could hopefully be utilized in further investigation of LTZ, especially PK/PD modeling and preclinical cancer research.
This study was funded by National Natural Science Foundation of China (Grant No. 81673500) andInnovation Team of Ministry of Education (Grant No. BMU2017TD003). We would like to express our heartfelt thanks to Dr. Jun Li (from State Key Laboratory of Natural and Biomimetic Drugs, Peking University) for kindly providing detailed and useful suggestions during the experiment.
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液相色谱-质谱串联定量测定裸鼠血浆中的来曲唑: 方法学建立、验证以及药物动力学研究应用
薛钧升2, 姚庆宇2, 李健2, 陈文君2, 苏红2, 田秀云3, 郝纯毅3, 周田彦1,2*
1. 北京大学医学部药学院天然药物及仿生药物国家重点实验室,北京100191
2. 北京大学医学部药学院药剂学系,北京100191
3. 北京大学肿瘤医院肝胆胰外二科北京大学肿瘤医院恶性肿瘤发病机制及转化研究教育部/北京市重点实验室,北京100142     
摘要: 本研究成功建立了一种灵敏、快速且简单的液相色谱-质谱串联方法, 用以测定裸鼠血浆中来曲唑的药物浓度, 并将其应用于药物动力学研究。以阿那曲唑作为内标, 血浆样本经过一步乙腈沉淀蛋白前处理后进行分析测定。采用C18反相柱(4.6 mm×250 mm, 5 μm), 乙腈–0.1%甲酸水溶液(60:40, v/v)为流动相组分, 流速1.0 mL/min, 以完成色谱分离过程。使用三重四极杆串联质谱, 以电喷雾正离子模式、质谱多反应监测技术对来曲唑和阿那曲唑同时进行质谱检测。标准曲线在0.8–2000 ng/mL浓度范围内呈现良好线性(r>0.99)。该方法定量下限可达0.8 ng/mL, 且日间、日内准确度、精密度均处于可接受范围。该方法成功应用于雌性BALB/c裸鼠单次口服来曲唑2 mg/kg的临床前药物动力学研究, 并建立一级吸收的一室模型以描述其药物动力学行为。 
关键词: 液质联用; 来曲唑; 裸鼠; 药物动力学 


Received: 2018-05-15, Revised: 2018-07-20, Accepted: 2018-09-13.
Foundation items: National Natural Science Foundation of China (Grant No. 81673500) and Innovation Team of Ministry of Education (Grant No. BMU2017TD003).
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