Determination of Gemcitabine in Plasma of Bladder Cancer Patients by Hydrophilic Interaction Chromatography with Ultraviolet Detection
Meng Wang1, Ryoichi Hiraki2, Norihiro Nakamura2, Tadashi Hayama3, Takuji Yasuda4, Yasuyoshi Miyata4, Hideki Sakai4, Mihoko N. Nakashima5, Mikiro Nakashima6, and Kaname Ohyama6,*
Abstract
Gemcitabine is a deoxycytidine analog that has been used for a broad spectrum of tumor, such as nonsmall-cell lung cancer, bladder cancer and pancreatic cancer. Because gemcitabine is hydrophilic, hydrophilic interaction liquid chromatography (HILIC), where analytes are retained on a polar column according to their hydrophilicity, should be adequate for separation analysis of gemcitabine. In the present study, we proposed a hydrophilic interaction chromatography with ultraviolet (HILIC-UV) method with liquid–liquid extraction and adding tetrahydrouridine to plasma samples for gemcitabine analysis of clinical samples with respect to daily and wide usage. The method successfully determined gemcitabine in 56 plasma samples of 30 unique patients. Mean plasma concentration of gemcitabine was 15.0 ± 6.0 μg/mL (mean ± standard deviation). The concentration range is consistent with the data from previous literatures. Our proposed HILIC- UV method is simple and easy handling, and is widely and clinically usable for determination of gemcitabine in human plasma.
Introduction
Gemcitabine is a deoxycytidine analog that has been used for a broad spectrum of tumor, such as non–small-cell lung cancer, bladder cancer and pancreatic cancer (1, 2). Several analytical methods have been developed for determination of gemcitabine, mainly reversed- phase liquid chromatography with ultraviolet (UV) detection (3) or with tandem mass spectrometry (MS/MS) (4). However, gemcitabine is hydrophilic; thus, it is weakly retained in reversed-phase liq- uid chromatography. Hydrophilic interaction liquid chromatography (HILIC), where analytes are retained on a polar column according to their hydrophilicity, should be more adequate for separation analysis of gemcitabine (5). Mano et al. has reported a hydrophilic interaction liquid chromatography with tandem mass spectrometry (HILIC- MS/MS) method for measuring gemcitabine in human plasma (6). This method has been fully validated in plasma samples but has yet been applied for real patients’ samples (6). In addition, MS/MS is still expensive, and has yet become widely used in clinical practice. In contrast, UV detection is not expensive and has an advantage in easier and wider use than mass spectrometry. In this study, we propose a hydrophilic interaction chromatography with ultravio- let (HILIC-UV) method with liquid–liquid extraction and adding tetrahydrouridine (to inhibit the conversion of gemcitabine to 2r,2r- difluoro-2r-deoxyuridine) to plasma samples for gemcitabine analysis of clinical samples with respect to daily and wide usage.
Experimental
Instrumentation and reagents
Ammonium acetate and isopropanol were purchased from Wako Pure Chemical Industries (Osaka, Japan). Acetonitrile and ethyl acetate were obtained from Nacalai Tesque (Kyoto, Japan). Gemc- itabine and 2r-deoxycytidine (internal standard, IS) were purchased from Tokyo Chemical Industry (Tokyo). The high-performance liquid chromatography was performed with a JASCO Pu-2080 Plus pump, UV-2075 plus detector and Chrom NAV software (Tokyo). The wavelength used for detecting gemcitabine and IS was set at 270 nm. The analytical column was SunShell HILIC-Amide (4.6 mm I.D. × 15 cm, particle size 2.6 μm, Tokyo). The column temperature was set at 30◦C. The mobile phase was 20 mM ammonium acetate in acetonitrile (acetonitrile/20 mM ammonium acetate = 90/10, v/v %). The flow rate was set at 1.0 mL/min. The injection volume was used in 20 μL.
Methods
Plasma samples (30 unique patients, 56 samples), from patients who received gemcitabine administration weekly for 3 weeks to treat bladder cancer, were collected at 30 min after infusion of gemcitabine (1000 mg/m2 [body surface area]). All experiments were performed in accordance with the Helsinki Declaration and with approval from the institutional ethics committees of Nagasaki University Hospital. Experiments were also performed with the written informed consent of the patients. The samples were collected into heparinized tubes containing tetrahydrouridine (0.15 mg/mL blood), which prevents ex vivo deamination and then, these samples were centrifuged at 1,200 g for 5 min. After that, transferred the supernatant into conical tubes and were kept in freezer at −80◦C.
A total of 10 μL of IS (100 μg/mL) was added into 190 μL of pool plasma or patients’ plasma samples. Then, 300 μL of isopropanol and 800 μL of ethyl acetate were added. The tubes vortexed for 20 sec and were centrifuged for 5 min at 2,800 g to pellet the precipitate. The supernatant fraction was filtered by disposable membrane (pore size 0.5 μm, ADVANTEC, Tokyo) into new clean conical centrifuge tubes and evaporated to dryness under vacuum distillation for 2 h at 50◦C. Then, the samples were reconstituted with 200 μL of the mixture of acetonitrile/20 mM ammonium acetate (95/5, v/v %), and were shaken vigorously, and were transferred into new tubes with membrane filtration.
Results
The retention times of gemcitabine and IS were measured with vary- ing mobile phase composition of acetonitrile and 20 mM ammonium acetate (90/10, 92/8, 95/5, v/v %). The separation was satisfactory at all concentrations and the fastest separation was achieved at the acetonitrile content of 90%. Calibration curves in human plasma were linear over the concentration range (0.5–50 μg/mL), based on the peak area ratio of gemcitabine to the IS. The correlation coefficient (r) was 0.999 (Supplementary Information S1). The recov- ery of gemcitabine from plasma was 78.1 (5 μg/mL) and 82.2% (25 μg/mL). The between-day (N = 3) repeatabilities (CV) calculated by analyzing a plasma sample including gemcitabine at 10 and 50 μg/mL were 7.4 and 2.0%, respectively. The developed HILIC- UV method was applied to the determination of gemcitabine in plasma samples from 30 patients (total 56 samples) receiving gemic- itabine therapy. The representative chromatograms obtained from blank plasma, spiked plasma (10 μg/mL) and a patient plasma are shown in Figure 1a–c. Measurement results of patients’ plasma sam- ples are shown in Table I. Gemcitabine concentrations of patients’ plasma ranged from 5.5 to 41.3 μg/mL. Mean plasma concentration of gemcitabine was 15.0 ± 6.0 μg/mL (mean ± standard devia- tion). The peak of gemcitabine could be detected in all the patient samples without interference from intrinsic components and co- administered drugs. Additionally, gemcitabine was clearly separated from the anticancer drug (cytarabine) containing similar structure with gemcitabine (Figure 1d). In order to determine the robustness of the method, analyzing a spiked plasma sample with gemcitabine (25 μg/mL) was performed before and after serial injections of plasma samples, and the peak area ratios of gemcitabine to the IS were 6.0 and 6.2, respectively.
Discussion
As expected, HILIC mode gave a sufficient separation of gemcitabine from intrinsic components of plasma in a short time (4 min). Analysis speed is not superior to previous HILIC-MS/MS method that detects gemcitabine within 2 min; however, the present method employs an inexpensive detection equipment and provided an enough sensitivity for clinical analysis of gemcitabine. Although the recovery of our method was around 80%, it was equal to the recoveries observed in previous studies (6, 7). In contrast to previous report developing HILIC-MS/MS method for determination of gemcitabine (6), our method was proved to successfully monitor the gemcitabine concen- tration in plasma samples of clinical patients receiving gemcitabine therapy. The concentration range of the patients is consistent with the data from previous literatures (8, 9). On the other hand, we evaluated the robustness of the method. After a total of 56 plasma sample injections, there was only 3% difference of the peak area ratio of gemcitabine to the IS between before and after the sequential analysis. Our proposed HILIC-UV method is simple and easy handling, and is widely and clinically usable for determination of gemcitabine in human plasma.
References
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