52 Original Article
Quantitative Determination of Fluconazole by UltraPerformance Liquid Chromatography Tandem Mass Spectrometry (UPLC-MS/MS) in Human Plasma and its Application to a Pharmacokinetic Study Authors
J.-j. Song1*, W. Li1*, Z. Wang1, D.-d. Tian1, W.-y. Yin2
Affiliations
1
Key words ▶ fluconazole ● ▶ UPLC-MS/MS ● ▶ plasma ● ▶ pharmacokinetic study ●
Abstract
Children’s Hospital of Zhengzhou, Zhengzhou Institute of Pediatric Research, Zhengzhou, China The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China
▼
In this study, a simple, rapid and sensitive ultraperformance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method is described for determination of fluconazole (FLA) in human plasma samples using phenacetin as the internal standard (IS). Sample preparation was accomplished through one-step protein pre cipitation by methanol, and chromatographic separation was performed on an Acquity BEH C18 column (2.1 mm × 50 mm, 1.7 μm) with mobile phase consisted of acetonitrile and water containing 0.1 % formic acid (40:60, v/v) at a flow
Introduction
▼ received 28.04.2014 accepted 07.07.2014 Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1384610 Published online: August 5, 2014 Drug Res 2015; 65: 52–56 © Georg Thieme Verlag KG Stuttgart · New York ISSN 2194-9379 Correspondence W. Yin The First Affiliated Hospital of Wenzhou Medical University Fuxue Lane 2 Lucheng District Wenzhou 325000 China Tel.: + 86/577/88879 444 Fax: + 86/577/88879 445 [email protected]
Fluconazole (FLA, ● ▶ Fig. 1) is an antifungal agent used in the treatment of oropharyngeal, eso phageal, or vulvovaginal candidiasis and in the treatment of other serious systemic candidial infections [1]. FLA is also a drug of choice for prophylaxis and treatment of systemic fungal infections in patients who are on immunosup pressive drug therapy [2]. FLA is administered orally once a day at a dose of 100–400 mg. More than 90 % of the drug is absorbed after oral administration. FLA has a long half-life and is excreted predominantly unchanged in urine [3]. A specific and sensitive analytical methodology is necessary to characterize the pharmacokinet ics of FLA and to determine the optimal dosing regimen for FLA in patients on immunosuppres sive drug therapy. Several methods have been reported for quantitative analysis of FLA in plasma and other biologic samples. Previous methods for the determination of FLA were based on high-performance liquid chromatography (HPLC) assay; the assay limit of quantification was not sufficiently low. Recent publications * These authors contributed equally to this work.
Song J-j et al. Determination of Fluconazole … Drug Res 2015; 65: 52–56
of 0.45 mL/min. Mass spectrometric analysis was performed using a QTrap 5500 mass spectrome ter coupled with an electro-spray ionization (ESI) source in the positive ion mode. The MRM transi tion of m/z 307.2→238.2 was used to quantify for FLA. The linearity of this method was found to be within the concentration range of 10–6 000 ng/ mL for FLA in human plasma. Only 1.0 min was needed for an analytical run. The method herein described was superior to previous methods and was successfully applied to the pharmacokinetic study of FLA in healthy Chinese volunteers after oral administration.
used a HPLC-ultraviolet method to measure FLA in human plasma; however, these methods require a liquid-liquid extraction procedure, use a large volume of plasma for extraction, or use high-salt content buffers in the mobile phase [4–6]. This potentially exposes individuals to organic solvents or requires additional washing time to clean the pump and column as a result of the use of buffers. Gas chromatography/mass spectrometry, liquid chromatography/mass spectrometry methods that are available are sen sitive, but the sample preparation is sometimes laborious [7–10]. There is no simple, specific, sensitive, and reproducible analytical method available to quantify FLA in human plasma. Ultra-performance liquid chromatography cou pled with tandem mass spectrometry (UPLC-MS/ MS) allows the selective and sensitive quantifica tion of structurally unrelated drugs in a single analytical run, resulting in substantial reductions in analytical time, turnaround time, and costs [11]. Therefore, it is worthwhile to develop a sim ple, precise, and accurate UPLC-MS/MS method for the determination of FLA in human plasma. The sample preparation of this method was sim ple one-step protein precipitation by methanol,
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2
Original Article 53 500, 1 000, 3 000 and 6 000 ng/mL for FLA. The preparation of QC samples were the same, with the 3 levels of plasma concentra tions (20, 1 000 and 4 800 ng/mL). In the same way, IS stock solu tion was made at an initial concentration of 1.00 mg/mL. The IS working solution (1.0 μg/mL) was made from the stock solution using methanol for dilution. All of the solutions were stored in a refrigerator at 4 °C.
Sample preparation
which was time- and effort-saving. The analysis time and the sensitivity could also meet the requirements of high-throughput bioanalysis. Once developed and validated, this method was suc cessfully applied to a pharmacokinetic study in healthy Chinese volunteers after oral administration of 300 mg FLA capsules.
Materials and Methods
▼
Chemicals and reagents
FLA (purity > 98.0 %) and phenacetin (purity > 98.0 %, IS) were both purchased from Sigma (St. Louis, MO, USA). LC-grade ace tonitrile and methanol were from Amethyst Chemicals. Blank human plasma was obtained from The First Affiliated Hospital of Wenzhou Medical University (Wenzhou, China). Ultra-pure water (resistance > 18 mΩ) was prepared by a Millipore Milli-Q purification system (Bedford, MA, USA).
Instrumentation and conditions
Liquid chromatography was performed on an Acquity ultra per formance liquid chromatography (UPLC) unit (Waters Corp., Mil ford, MA) with an Acquity BEH C18 column (2.1 mm × 50 mm, 1.7 μm particle size) and inline 0.2 μm stainless steel frit filter (Waters Corp., Milford, USA). The mobile phase consisted of ace tonitrile and water containing 0.1 % formic acid (40:60, v/v). The flow rate was 0.45 mL/min. The overall run time was 1.0 min. An AB Sciex QTRAP 5500 triple quadrupole mass spectrometer equipped with an electro-spray ionization (ESI) source (Toronto, Canada) was used for mass spectrometric detection. The detec tion was operated in the multiple reaction monitoring (MRM) mode under unit mass resolution (0.7 amu) in mass analyzers. The dwell time was set to 250 ms for each MRM transition. The MRM transitions were m/z 307.2 → 238.2 and m/z 180.0 → 138.0 for FLA and IS, respectively. After optimization, the source parameters were set as follows: curtain gas, 35 psig; nebulizer gas, 50 psig; turbo gas, 60 psig; ion spray voltage, 4.0 kV; and temperature, 300 °C. Data acquiring and processing were per formed using analyst software (version 1.5, AB Sciex).
Preparation of standard and quality control (QC) samples
The stock solution of FLA that was used to make the calibration standards and quality control (QC) samples was prepared by dis solving 10.00 mg in 10 mL methanol to obtain a concentration of 1.00 mg/mL. The working solutions for calibration and quality controls were made from the stock solution by diluting with methanol. Calibration curve standards were prepared by spiking blank human plasma with appropriate amounts of the working solutions at final drug concentrations of 10, 20, 50, 100, 200,
Before analysis, the plasma sample was thawed to room tem perature. In a 1.5 mL centrifuge tube, an aliquot of 200 µL of the IS working solution (1.0 μg/mL in methanol) was added to 100 µL of collected plasma sample. The tubes were vortex mixed for 1.0 min and spun in a centrifuge at 12 000 rmp for 10 min. The supernatant (20 µL) was injected into the UPLC-MS/MS system for analysis.
Method validation
Specificity was determined by analysis of blank human plasma samples from 6 different volunteers, every blank sample was handled by the procedure described in “Sample preparation” and confirmed that endogenous substances did not have the possible interference with the analyte and the IS. Plasma samples were quantified using the calibration curve. Calibration curves were constructed validated by analyzing spiked calibration samples on 3 days in a row. Peak area ratio of FLA to IS was plotted against analyte concentrations, and stand ard curves were fitted by weighted (1/χ2) least squares linear regression in the concentration of 10–6 000 ng/mL for FLA. A cor relation of more than 0.99 was desirable for all the calibration curves. The sensitivity of the method was determined by quan tifying the lower limit of quantification (LLOQ). The LLOQ was defined as the lowest acceptable point in the calibration curve which were determined at an acceptable precision and accuracy. To determine the matrix effect, 6 different blank plasma samples were utilized to prepare QC samples and used for assessing the lot-to-lot matrix effect. Matrix effect was evaluated at 3 QC lev els by comparing the peak areas of the analyte obtained from plasma samples spiked with the analyte after extraction to those of the pure standard solutions at the same concentrations. The matrix effect of IS was evaluated at the working concentration (1.0 μg/mL) in the same manner. The extraction recoveries of FLA at 3 QC levels (n = 6) were deter mined by comparing peak area of the analyte in samples that were spiked with the analyte prior to extraction with those of samples to which the corresponding solution was added after extraction. The extraction recovery of the IS at the working concentration (1.0 μg/mL) was determined in a similar way as a reference. The intra-day precision and accuracy of FLA were evaluated by analyzing QC samples (20, 1 000 and 4 800 ng/mL) with 6 repli cates for each concentration. The inter-day precision and accu racy were evaluated by analyzing QC samples with 6 replicates for each concentration over 6 days. The precision was expressed by relative standard deviation (RSD) and the accuracy by relative error (RE). The stabilities of FLA in human plasma were tested by analyzing 5 replicates of plasma samples at 3 concentration levels (20, 1 000 and 4 800 ng/mL) in different conditions. The short-term stability was determined after the exposure of the spiked samples at room temperature for 2 h, and the ready-to-inject samples (after extraction) in the autosampler at 4 °C for 12 h. The freeze-thaw stability was evaluated after 3 complete freezeSong J-j et al. Determination of Fluconazole … Drug Res 2015; 65: 52–56
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Fig. 1 The chemical structures of FLA a and phenacetin (IS, b).
54 Original Article
Pharmacokinetic study
The clinical protocol was approved by Medical Ethics Committee of the First Affiliated Hospital of Wenzhou Medical University prior to the study. 20 volunteers were given written informed consent to participate in the study according to the principles of the Declaration of Helsinki. The volunteers who submitted the agreements to attend this project were medically examined for the pharmacokinetics study of FLA. The subjects were required to abstain from taking any other drug for 7 days prior to the start of test. They were also demanded not to smoke or drink alcohol for 24 h before the beginning of the study until its end. All volun teers were received an oral dose of 300 mg FLA capsules with 200 mL water. Blood samples (3 mL) were collected into heparin ized tubes at 0.5, 1, 2, 3, 4, 8, 12, 24, 48, 72, 96 and 120 h after oral administration. Blood samples were centrifuged at 4 000 × g for 10 min and the plasma was separated and kept frozen at − 20 °C until analysis.
Data analysis
Plasma concentration vs. time profiles were analyzed using DAS software (Version 2.0, Medical University of Wenzhou, China) to estimate the type of compartment model and pharmacokinetic parameters. Data were expressed as mean ± SD.
Results and Discussion
▼
Method development
Owing to the complex matrices, sample preparation is usually required for the determination of the analytes in biological sam ples in order to remove the possible interfering matrix compo nents and increase selectivity and sensitivity. Compared to liquid-liquid extraction for the sample preparation, organic solvent precipitation was used for the sample preparation. This
simple procedure produced a clean chromatogram for the blank plasma sample and yielded satisfactory recoveries for the ana lytes. In this work, 200 µL of methanol was added in the process of sample preparation. Internal standard plays an important role in biopharmaceutical analysis and is often required to have sim ilar physical and chemical properties to analyte in terms of solu bility and acid-base properties. On the basis of the above requirements, phenacetin was found to be suitable for the pre sent work and finally used as the internal standard. Other chro matographic conditions, especially the composition of mobile phase, were tested to achieve good resolution and symmetric peak shapes of analytes as well as a short run time. It was found that acetonitrile and water containing 0.1 % formic acid (40:60, v/v) could achieve our purpose and were finally adopted as the mobile phase for chromatographic separation. The retention time was 0.82 min for FLA and 0.56 min for IS. The run time was less than 1.0 min. The advantage of this method is that a rela tively larger number of samples can be analyzed in a short time, thus increasing output.
Specificity
UPLC chromatograms of human plasma showed that the reten tion times for FLA and IS were approximately 0.82 and 0.56 min, respectively. ● ▶ Fig. 2 shows the typical chromatograms of a blank plasma sample, a blank plasma sample spiked with FLA and IS, and a plasma sample from a healthy volunteer after an oral administration. No endogenous interferences were observed in the blank plasma samples for the analyte.
Linearity of calibration curve and sensitivity
The linear regressions of the peak area ratios vs. concentrations were fitted over the concentration range of 10–6 000 ng/mL for FLA in human plasma. The typical equations of the calibration curve was as follows: y = 6.2145x − 0.1524, r = 0.9997, where y represents the ratio of peak area to that of IS, and x represents the plasma concentration. The LLOQ was estimated in the p rocess of calibration curve construction and defined as the concentration giving a signal-noise ratio of 10, was 10 ng/mL for FLA.
Fig. 2 Representative UPLC-MS/MS chromatograms for FLA and phenacetin (IS) in human plasma samples: a blank plasma sample; b blank plasma sample spiked with FLA and IS; c human plasma sample 1.0 h after oral administration of single dosage 300 mg FLA.
Song J-j et al. Determination of Fluconazole … Drug Res 2015; 65: 52–56
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thaw cycles ( − 20 to 25 °C) on consecutive days. The long-term stability was assessed after storage of the standard spiked plasma samples at − 20 °C for 80 days.
Original Article 55 less, and the inter-day precision was 9.8 % or less at each QC level. The accuracy of the method ranged from − 8.6 to 9.5 % for FLA at 3 QC levels. Assay performance data is presented in ● ▶ Table 2. The above results demonstrated that the values were within the acceptable range and the method was accurate and precise.
To avoid interference from exogenous compounds co-eluted with the target compound, MS/MS detection, offering unique selectivity against matrix background and requires very limited sample preparation was performed. Ionization of the analyte was carried out using the ESI technique with positive polarity and MRM mode. The matrix effect on the ionization efficiency of the analyte was evaluated by comparing the peak response of the analyte dissolved in blank sample extract with those for the analyte dissolved to the same concentrations in methanol. The matrix effect for FLA at concentrations of 20, 1 000 and 4 800 ng/ mL and IS (1.0 μg/mL) were showed in ● ▶ Table 1. As a result, matrix effect from plasma was negligible in this method. The recovery was calculated by comparing the mean peak areas of the analyte spiked before extraction divided by the areas of the analyte samples spiked after extraction and multiplied by 100 %. Results are shown in ● ▶ Table 1. The recovery in plasma ranged from 84.5 to 86.5 % for FLA. The recovery of IS (1.0 μg/mL) in plasma was 84.2 %.
All the stability studies of FLA in plasma were conducted at 3 concentration levels (20, 1 000 and 4 800 ng/mL) with 5 determi nations for each under different storage conditions. The RSD of the mean test responses was within 10 % in all stability tests of FLA in plasma. The results of stability experiments are summa rized in ● ▶ Table 3. No effect on the quantitation was observed for plasma samples kept at room temperature for 2 h and at 4 °C for 12 h in an autosa mpler. There was also no significant degradation when samples of FLA in plasma were taken through 3 freeze ( − 20 °C)-thaw (25 °C) cycles. And it was also stable at − 20 °C for 80 days.
Precision and accuracy
Application of the method
The precision of the method was evaluated by calculating RSD for QCs at 3 concentration levels (20, 1 000 and 4 800 ng/mL) over 3 validation days. The intra-day precision for FLA was 8.5 % or Table 1 Matrix effect and Recovery of FLA and internal standards from human plasma (n = 6). Ana-
Concentra-
Matrix effect ( %)
Recovery ( %)
lytes
tion added
Mean ± SD
Mean ± SD
(ng/mL) FLA IS
20 1 000 4 800 1 000
RSD
RSD
( %) 95.4 ± 6.9 94.5 ± 4.5 96.5 ± 6.6 94.2 ± 7.7
7.9 4.8 6.8 8.2
( %) 85.4 ± 6.9 84.5 ± 4.5 86.5 ± 6.6 84.2 ± 7.7
8.1 5.4 7.7 9.2
Stability
This validated UPLC-MS/MS method was successfully applied to a pharmacokinetic study of FLA in 20 Chinese healthy male vol unteers after administration of 300 mg capsules. The profile of the mean plasma concentration-time curve of FLA in single dose study is shown in ● ▶ Fig. 3. After administration of a single dose of 300 mg FLA capsules, the Cmax and Tmax were 5.76 ± 0.75 μg/mL and 2.50 ± 0.89 h, respec tively. Plasma concentration declined with a t1/2 of 29.60 ± 4.98 h. The AUC0–120 and AUC0→∞ values obtained were 224.60 ± 28.25 and 241.40 ± 32.37 μg/mL · h, respectively. Results are shown in ● ▶ Table 4.
Conclusions
▼ Table 2 Precision and Accuracy for FLA of QC samples in human plasma (n = 6).
Compound FLA
Concen-
RSD %
RE %
tration (ng/mL) 20 1 000 4 800
intra-day 8.5 6.5 3.5
inter-day 9.8 8.3 4.6
intra-day
inter-day
− 8.6 − 7.8 3.6
9.5 8.4 5.5
A rapid, sensitive and selective UPLC-MS/MS method for the determination of FLA in human plasma was developed and vali dated. Compared with the analytical methods reported in the literatures, the method offered superior sample preparation with a simple one-step protein precipitation by methanol and shorter run time of 1.0 min. The method meets the requirement of high sample throughput in bioanalysis and has been success fully applied to the pharmacokinetic study of FLA capsule in healthy volunteers.
Table 3 Stability of FLA under various storage conditions (n = 5). Storage conditions room temperature/2 h
4 °C/12 h
− 20 °C/3 freeze-thaw cycles
− 20 °C/80 days
Concentration added (ng/mL) 20 1 000 4 800 20 1 000 4 800 20 1 000 4 800 20 1 000 4 800
Concentration measured (ng/mL) 21.48 ± 1.82 1 064.98 ± 72.33 4 285.43 ± 326.85 22.54 ± 1.28 1 033.51 ± 80.38 4 578.43 ± 333.58 19.65 ± 1.79 1 111.01 ± 98.77 4 789.36 ± 132.11 20.96 ± 1.64 911.24 ± 56.39 4 899.76 ± 256.36
RSD % 8.5 6.8 7.6 5.7 7.8 7.3 9.1 8.9 2.8 7.8 6.2 5.2
RE % 6.8 3.5 − 4.5 6.5 5.4 − 6.1 − 3.3 7.5 − 2.1 2.3 − 5.6 4.8
Song J-j et al. Determination of Fluconazole … Drug Res 2015; 65: 52–56
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Matrix effect and recovery
56 Original Article
Fig. 3 Mean plasma concentration of FLA after oral administration of single dose of 300 mg of FLA to 20 healthy human subjects under fasting condition.
Table 4 Main pharmacokinetic parameters of FLA in 20 healthy volunteers after oral administration 300 mg to 20 Chinese healthy male volunteers. Parameter t1/2 (h) Cmax (μg/mL) Tmax (h) AUC0→120 (μg/mL · h) AUC0→∞ (μg/mL · h)
Fluconazole 29.60 ± 4.98 5.76 ± 0.75 2.50 ± 0.89 224.60 ± 28.25 241.40 ± 32.37
Declaration of Interest
▼
The authors report no conflicts of interest.
Song J-j et al. Determination of Fluconazole … Drug Res 2015; 65: 52–56
1 Rosa MI, Silva BR, Pires PS et al. Weekly fluconazole therapy for recur rent vulvovaginal candidiasis: a systematic review and meta-analysis. Eur J Obstet Gynecol Reprod Biol 2013; 167: 132–136 2 Ethier MC, Science M, Beyene J et al. Mould-active compared with flu conazole prophylaxis to prevent invasive fungal diseases in cancer patients receiving chemotherapy or haematopoietic stem-cell trans plantation: a systematic review and meta-analysis of randomised controlled trials. Br J Cancer 2012; 106: 1626–1637 3 Humphrey MJ, Jevons S, Tarbit MH. Pharmacokinetic evaluation of UK-49,858, a metabolically stable triazole antifungal drug, in animals and humans. Antimicrob Agents Chemother 1985; 28: 648–653 4 Kim SS, Im HT, Kang IM et al. An optimized analytical method of fluconazole in human plasma by high-performance liquid chroma tography with ultraviolet detection and its application to a bioequiva lence study. J Chromatogr B Analyt Technol Biomed Life Sci 2007; 852: 174–179 5 Wattananat T, Akarawut W. Validated HPLC method for the determi nation of fluconazole in human plasma. Biomed Chromatogr 2006; 20: 1–3 6 Belal F, Sharaf El-Din MK, Eid MI et al. Micellar HPLC and Derivative Spectrophotometric Methods for the Simultaneous Determination of Fluconazole and Tinidazole in Pharmaceuticals and Biological Fluids. J Chromatogr Sci 2013 7 Rege AB, Walker-Cador JY, Clark RA et al. Rapid and sensitive assay for fluconazole which uses gas chromatography with electron capture detection. Antimicrob Agents Chemother 1992; 36: 647–650 8 Decosterd LA, Rochat B, Pesse B et al. Multiplex ultra-performance liq uid chromatography-tandem mass spectrometry method for simul taneous quantification in human plasma of fluconazole, itraconazole, hydroxyitraconazole, posaconazole, voriconazole, voriconazole-Noxide, anidulafungin, and caspofungin. Antimicrob Agents Chemother 2010; 54: 5303–5315 9 Wu D, Wade KC, Paul DJ et al. A rapid and sensitive LC-MS/MS method for determination of fluconazole in human plasma and its applica tion in infants with Candida infections. Ther Drug Monit 2009; 31: 703–709 10 Wood PR, Tarbit MH. Gas chromatographic method for the determina tion of fluconazole, a novel antifungal agent, in human plasma and urine. J Chromatogr 1986; 383: 179–186 11 Toyo’oka T. Determination methods for biologically active compounds by ultra-performance liquid chromatography coupled with mass spectrometry: application to the analyses of pharmaceuticals, foods, plants, environments, metabonomics, and metabolomics. J Chroma togr Sci 2008; 46: 233–247
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References
Quantitative Determination of Fluconazole by UltraPerformance Liquid Chromatography Tandem Mass Spectrometry (UPLC-MS/MS) in Human Plasma and its Application to a Pharmacokinetic Study Authors
J.-j. Song1*, W. Li1*, Z. Wang1, D.-d. Tian1, W.-y. Yin2
Affiliations
1
Key words ▶ fluconazole ● ▶ UPLC-MS/MS ● ▶ plasma ● ▶ pharmacokinetic study ●
Abstract
Children’s Hospital of Zhengzhou, Zhengzhou Institute of Pediatric Research, Zhengzhou, China The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China
▼
In this study, a simple, rapid and sensitive ultraperformance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method is described for determination of fluconazole (FLA) in human plasma samples using phenacetin as the internal standard (IS). Sample preparation was accomplished through one-step protein pre cipitation by methanol, and chromatographic separation was performed on an Acquity BEH C18 column (2.1 mm × 50 mm, 1.7 μm) with mobile phase consisted of acetonitrile and water containing 0.1 % formic acid (40:60, v/v) at a flow
Introduction
▼ received 28.04.2014 accepted 07.07.2014 Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1384610 Published online: August 5, 2014 Drug Res 2015; 65: 52–56 © Georg Thieme Verlag KG Stuttgart · New York ISSN 2194-9379 Correspondence W. Yin The First Affiliated Hospital of Wenzhou Medical University Fuxue Lane 2 Lucheng District Wenzhou 325000 China Tel.: + 86/577/88879 444 Fax: + 86/577/88879 445 [email protected]
Fluconazole (FLA, ● ▶ Fig. 1) is an antifungal agent used in the treatment of oropharyngeal, eso phageal, or vulvovaginal candidiasis and in the treatment of other serious systemic candidial infections [1]. FLA is also a drug of choice for prophylaxis and treatment of systemic fungal infections in patients who are on immunosup pressive drug therapy [2]. FLA is administered orally once a day at a dose of 100–400 mg. More than 90 % of the drug is absorbed after oral administration. FLA has a long half-life and is excreted predominantly unchanged in urine [3]. A specific and sensitive analytical methodology is necessary to characterize the pharmacokinet ics of FLA and to determine the optimal dosing regimen for FLA in patients on immunosuppres sive drug therapy. Several methods have been reported for quantitative analysis of FLA in plasma and other biologic samples. Previous methods for the determination of FLA were based on high-performance liquid chromatography (HPLC) assay; the assay limit of quantification was not sufficiently low. Recent publications * These authors contributed equally to this work.
Song J-j et al. Determination of Fluconazole … Drug Res 2015; 65: 52–56
of 0.45 mL/min. Mass spectrometric analysis was performed using a QTrap 5500 mass spectrome ter coupled with an electro-spray ionization (ESI) source in the positive ion mode. The MRM transi tion of m/z 307.2→238.2 was used to quantify for FLA. The linearity of this method was found to be within the concentration range of 10–6 000 ng/ mL for FLA in human plasma. Only 1.0 min was needed for an analytical run. The method herein described was superior to previous methods and was successfully applied to the pharmacokinetic study of FLA in healthy Chinese volunteers after oral administration.
used a HPLC-ultraviolet method to measure FLA in human plasma; however, these methods require a liquid-liquid extraction procedure, use a large volume of plasma for extraction, or use high-salt content buffers in the mobile phase [4–6]. This potentially exposes individuals to organic solvents or requires additional washing time to clean the pump and column as a result of the use of buffers. Gas chromatography/mass spectrometry, liquid chromatography/mass spectrometry methods that are available are sen sitive, but the sample preparation is sometimes laborious [7–10]. There is no simple, specific, sensitive, and reproducible analytical method available to quantify FLA in human plasma. Ultra-performance liquid chromatography cou pled with tandem mass spectrometry (UPLC-MS/ MS) allows the selective and sensitive quantifica tion of structurally unrelated drugs in a single analytical run, resulting in substantial reductions in analytical time, turnaround time, and costs [11]. Therefore, it is worthwhile to develop a sim ple, precise, and accurate UPLC-MS/MS method for the determination of FLA in human plasma. The sample preparation of this method was sim ple one-step protein precipitation by methanol,
Downloaded by: East Carolina University. Copyrighted material.
2
Original Article 53 500, 1 000, 3 000 and 6 000 ng/mL for FLA. The preparation of QC samples were the same, with the 3 levels of plasma concentra tions (20, 1 000 and 4 800 ng/mL). In the same way, IS stock solu tion was made at an initial concentration of 1.00 mg/mL. The IS working solution (1.0 μg/mL) was made from the stock solution using methanol for dilution. All of the solutions were stored in a refrigerator at 4 °C.
Sample preparation
which was time- and effort-saving. The analysis time and the sensitivity could also meet the requirements of high-throughput bioanalysis. Once developed and validated, this method was suc cessfully applied to a pharmacokinetic study in healthy Chinese volunteers after oral administration of 300 mg FLA capsules.
Materials and Methods
▼
Chemicals and reagents
FLA (purity > 98.0 %) and phenacetin (purity > 98.0 %, IS) were both purchased from Sigma (St. Louis, MO, USA). LC-grade ace tonitrile and methanol were from Amethyst Chemicals. Blank human plasma was obtained from The First Affiliated Hospital of Wenzhou Medical University (Wenzhou, China). Ultra-pure water (resistance > 18 mΩ) was prepared by a Millipore Milli-Q purification system (Bedford, MA, USA).
Instrumentation and conditions
Liquid chromatography was performed on an Acquity ultra per formance liquid chromatography (UPLC) unit (Waters Corp., Mil ford, MA) with an Acquity BEH C18 column (2.1 mm × 50 mm, 1.7 μm particle size) and inline 0.2 μm stainless steel frit filter (Waters Corp., Milford, USA). The mobile phase consisted of ace tonitrile and water containing 0.1 % formic acid (40:60, v/v). The flow rate was 0.45 mL/min. The overall run time was 1.0 min. An AB Sciex QTRAP 5500 triple quadrupole mass spectrometer equipped with an electro-spray ionization (ESI) source (Toronto, Canada) was used for mass spectrometric detection. The detec tion was operated in the multiple reaction monitoring (MRM) mode under unit mass resolution (0.7 amu) in mass analyzers. The dwell time was set to 250 ms for each MRM transition. The MRM transitions were m/z 307.2 → 238.2 and m/z 180.0 → 138.0 for FLA and IS, respectively. After optimization, the source parameters were set as follows: curtain gas, 35 psig; nebulizer gas, 50 psig; turbo gas, 60 psig; ion spray voltage, 4.0 kV; and temperature, 300 °C. Data acquiring and processing were per formed using analyst software (version 1.5, AB Sciex).
Preparation of standard and quality control (QC) samples
The stock solution of FLA that was used to make the calibration standards and quality control (QC) samples was prepared by dis solving 10.00 mg in 10 mL methanol to obtain a concentration of 1.00 mg/mL. The working solutions for calibration and quality controls were made from the stock solution by diluting with methanol. Calibration curve standards were prepared by spiking blank human plasma with appropriate amounts of the working solutions at final drug concentrations of 10, 20, 50, 100, 200,
Before analysis, the plasma sample was thawed to room tem perature. In a 1.5 mL centrifuge tube, an aliquot of 200 µL of the IS working solution (1.0 μg/mL in methanol) was added to 100 µL of collected plasma sample. The tubes were vortex mixed for 1.0 min and spun in a centrifuge at 12 000 rmp for 10 min. The supernatant (20 µL) was injected into the UPLC-MS/MS system for analysis.
Method validation
Specificity was determined by analysis of blank human plasma samples from 6 different volunteers, every blank sample was handled by the procedure described in “Sample preparation” and confirmed that endogenous substances did not have the possible interference with the analyte and the IS. Plasma samples were quantified using the calibration curve. Calibration curves were constructed validated by analyzing spiked calibration samples on 3 days in a row. Peak area ratio of FLA to IS was plotted against analyte concentrations, and stand ard curves were fitted by weighted (1/χ2) least squares linear regression in the concentration of 10–6 000 ng/mL for FLA. A cor relation of more than 0.99 was desirable for all the calibration curves. The sensitivity of the method was determined by quan tifying the lower limit of quantification (LLOQ). The LLOQ was defined as the lowest acceptable point in the calibration curve which were determined at an acceptable precision and accuracy. To determine the matrix effect, 6 different blank plasma samples were utilized to prepare QC samples and used for assessing the lot-to-lot matrix effect. Matrix effect was evaluated at 3 QC lev els by comparing the peak areas of the analyte obtained from plasma samples spiked with the analyte after extraction to those of the pure standard solutions at the same concentrations. The matrix effect of IS was evaluated at the working concentration (1.0 μg/mL) in the same manner. The extraction recoveries of FLA at 3 QC levels (n = 6) were deter mined by comparing peak area of the analyte in samples that were spiked with the analyte prior to extraction with those of samples to which the corresponding solution was added after extraction. The extraction recovery of the IS at the working concentration (1.0 μg/mL) was determined in a similar way as a reference. The intra-day precision and accuracy of FLA were evaluated by analyzing QC samples (20, 1 000 and 4 800 ng/mL) with 6 repli cates for each concentration. The inter-day precision and accu racy were evaluated by analyzing QC samples with 6 replicates for each concentration over 6 days. The precision was expressed by relative standard deviation (RSD) and the accuracy by relative error (RE). The stabilities of FLA in human plasma were tested by analyzing 5 replicates of plasma samples at 3 concentration levels (20, 1 000 and 4 800 ng/mL) in different conditions. The short-term stability was determined after the exposure of the spiked samples at room temperature for 2 h, and the ready-to-inject samples (after extraction) in the autosampler at 4 °C for 12 h. The freeze-thaw stability was evaluated after 3 complete freezeSong J-j et al. Determination of Fluconazole … Drug Res 2015; 65: 52–56
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Fig. 1 The chemical structures of FLA a and phenacetin (IS, b).
54 Original Article
Pharmacokinetic study
The clinical protocol was approved by Medical Ethics Committee of the First Affiliated Hospital of Wenzhou Medical University prior to the study. 20 volunteers were given written informed consent to participate in the study according to the principles of the Declaration of Helsinki. The volunteers who submitted the agreements to attend this project were medically examined for the pharmacokinetics study of FLA. The subjects were required to abstain from taking any other drug for 7 days prior to the start of test. They were also demanded not to smoke or drink alcohol for 24 h before the beginning of the study until its end. All volun teers were received an oral dose of 300 mg FLA capsules with 200 mL water. Blood samples (3 mL) were collected into heparin ized tubes at 0.5, 1, 2, 3, 4, 8, 12, 24, 48, 72, 96 and 120 h after oral administration. Blood samples were centrifuged at 4 000 × g for 10 min and the plasma was separated and kept frozen at − 20 °C until analysis.
Data analysis
Plasma concentration vs. time profiles were analyzed using DAS software (Version 2.0, Medical University of Wenzhou, China) to estimate the type of compartment model and pharmacokinetic parameters. Data were expressed as mean ± SD.
Results and Discussion
▼
Method development
Owing to the complex matrices, sample preparation is usually required for the determination of the analytes in biological sam ples in order to remove the possible interfering matrix compo nents and increase selectivity and sensitivity. Compared to liquid-liquid extraction for the sample preparation, organic solvent precipitation was used for the sample preparation. This
simple procedure produced a clean chromatogram for the blank plasma sample and yielded satisfactory recoveries for the ana lytes. In this work, 200 µL of methanol was added in the process of sample preparation. Internal standard plays an important role in biopharmaceutical analysis and is often required to have sim ilar physical and chemical properties to analyte in terms of solu bility and acid-base properties. On the basis of the above requirements, phenacetin was found to be suitable for the pre sent work and finally used as the internal standard. Other chro matographic conditions, especially the composition of mobile phase, were tested to achieve good resolution and symmetric peak shapes of analytes as well as a short run time. It was found that acetonitrile and water containing 0.1 % formic acid (40:60, v/v) could achieve our purpose and were finally adopted as the mobile phase for chromatographic separation. The retention time was 0.82 min for FLA and 0.56 min for IS. The run time was less than 1.0 min. The advantage of this method is that a rela tively larger number of samples can be analyzed in a short time, thus increasing output.
Specificity
UPLC chromatograms of human plasma showed that the reten tion times for FLA and IS were approximately 0.82 and 0.56 min, respectively. ● ▶ Fig. 2 shows the typical chromatograms of a blank plasma sample, a blank plasma sample spiked with FLA and IS, and a plasma sample from a healthy volunteer after an oral administration. No endogenous interferences were observed in the blank plasma samples for the analyte.
Linearity of calibration curve and sensitivity
The linear regressions of the peak area ratios vs. concentrations were fitted over the concentration range of 10–6 000 ng/mL for FLA in human plasma. The typical equations of the calibration curve was as follows: y = 6.2145x − 0.1524, r = 0.9997, where y represents the ratio of peak area to that of IS, and x represents the plasma concentration. The LLOQ was estimated in the p rocess of calibration curve construction and defined as the concentration giving a signal-noise ratio of 10, was 10 ng/mL for FLA.
Fig. 2 Representative UPLC-MS/MS chromatograms for FLA and phenacetin (IS) in human plasma samples: a blank plasma sample; b blank plasma sample spiked with FLA and IS; c human plasma sample 1.0 h after oral administration of single dosage 300 mg FLA.
Song J-j et al. Determination of Fluconazole … Drug Res 2015; 65: 52–56
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thaw cycles ( − 20 to 25 °C) on consecutive days. The long-term stability was assessed after storage of the standard spiked plasma samples at − 20 °C for 80 days.
Original Article 55 less, and the inter-day precision was 9.8 % or less at each QC level. The accuracy of the method ranged from − 8.6 to 9.5 % for FLA at 3 QC levels. Assay performance data is presented in ● ▶ Table 2. The above results demonstrated that the values were within the acceptable range and the method was accurate and precise.
To avoid interference from exogenous compounds co-eluted with the target compound, MS/MS detection, offering unique selectivity against matrix background and requires very limited sample preparation was performed. Ionization of the analyte was carried out using the ESI technique with positive polarity and MRM mode. The matrix effect on the ionization efficiency of the analyte was evaluated by comparing the peak response of the analyte dissolved in blank sample extract with those for the analyte dissolved to the same concentrations in methanol. The matrix effect for FLA at concentrations of 20, 1 000 and 4 800 ng/ mL and IS (1.0 μg/mL) were showed in ● ▶ Table 1. As a result, matrix effect from plasma was negligible in this method. The recovery was calculated by comparing the mean peak areas of the analyte spiked before extraction divided by the areas of the analyte samples spiked after extraction and multiplied by 100 %. Results are shown in ● ▶ Table 1. The recovery in plasma ranged from 84.5 to 86.5 % for FLA. The recovery of IS (1.0 μg/mL) in plasma was 84.2 %.
All the stability studies of FLA in plasma were conducted at 3 concentration levels (20, 1 000 and 4 800 ng/mL) with 5 determi nations for each under different storage conditions. The RSD of the mean test responses was within 10 % in all stability tests of FLA in plasma. The results of stability experiments are summa rized in ● ▶ Table 3. No effect on the quantitation was observed for plasma samples kept at room temperature for 2 h and at 4 °C for 12 h in an autosa mpler. There was also no significant degradation when samples of FLA in plasma were taken through 3 freeze ( − 20 °C)-thaw (25 °C) cycles. And it was also stable at − 20 °C for 80 days.
Precision and accuracy
Application of the method
The precision of the method was evaluated by calculating RSD for QCs at 3 concentration levels (20, 1 000 and 4 800 ng/mL) over 3 validation days. The intra-day precision for FLA was 8.5 % or Table 1 Matrix effect and Recovery of FLA and internal standards from human plasma (n = 6). Ana-
Concentra-
Matrix effect ( %)
Recovery ( %)
lytes
tion added
Mean ± SD
Mean ± SD
(ng/mL) FLA IS
20 1 000 4 800 1 000
RSD
RSD
( %) 95.4 ± 6.9 94.5 ± 4.5 96.5 ± 6.6 94.2 ± 7.7
7.9 4.8 6.8 8.2
( %) 85.4 ± 6.9 84.5 ± 4.5 86.5 ± 6.6 84.2 ± 7.7
8.1 5.4 7.7 9.2
Stability
This validated UPLC-MS/MS method was successfully applied to a pharmacokinetic study of FLA in 20 Chinese healthy male vol unteers after administration of 300 mg capsules. The profile of the mean plasma concentration-time curve of FLA in single dose study is shown in ● ▶ Fig. 3. After administration of a single dose of 300 mg FLA capsules, the Cmax and Tmax were 5.76 ± 0.75 μg/mL and 2.50 ± 0.89 h, respec tively. Plasma concentration declined with a t1/2 of 29.60 ± 4.98 h. The AUC0–120 and AUC0→∞ values obtained were 224.60 ± 28.25 and 241.40 ± 32.37 μg/mL · h, respectively. Results are shown in ● ▶ Table 4.
Conclusions
▼ Table 2 Precision and Accuracy for FLA of QC samples in human plasma (n = 6).
Compound FLA
Concen-
RSD %
RE %
tration (ng/mL) 20 1 000 4 800
intra-day 8.5 6.5 3.5
inter-day 9.8 8.3 4.6
intra-day
inter-day
− 8.6 − 7.8 3.6
9.5 8.4 5.5
A rapid, sensitive and selective UPLC-MS/MS method for the determination of FLA in human plasma was developed and vali dated. Compared with the analytical methods reported in the literatures, the method offered superior sample preparation with a simple one-step protein precipitation by methanol and shorter run time of 1.0 min. The method meets the requirement of high sample throughput in bioanalysis and has been success fully applied to the pharmacokinetic study of FLA capsule in healthy volunteers.
Table 3 Stability of FLA under various storage conditions (n = 5). Storage conditions room temperature/2 h
4 °C/12 h
− 20 °C/3 freeze-thaw cycles
− 20 °C/80 days
Concentration added (ng/mL) 20 1 000 4 800 20 1 000 4 800 20 1 000 4 800 20 1 000 4 800
Concentration measured (ng/mL) 21.48 ± 1.82 1 064.98 ± 72.33 4 285.43 ± 326.85 22.54 ± 1.28 1 033.51 ± 80.38 4 578.43 ± 333.58 19.65 ± 1.79 1 111.01 ± 98.77 4 789.36 ± 132.11 20.96 ± 1.64 911.24 ± 56.39 4 899.76 ± 256.36
RSD % 8.5 6.8 7.6 5.7 7.8 7.3 9.1 8.9 2.8 7.8 6.2 5.2
RE % 6.8 3.5 − 4.5 6.5 5.4 − 6.1 − 3.3 7.5 − 2.1 2.3 − 5.6 4.8
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Matrix effect and recovery
56 Original Article
Fig. 3 Mean plasma concentration of FLA after oral administration of single dose of 300 mg of FLA to 20 healthy human subjects under fasting condition.
Table 4 Main pharmacokinetic parameters of FLA in 20 healthy volunteers after oral administration 300 mg to 20 Chinese healthy male volunteers. Parameter t1/2 (h) Cmax (μg/mL) Tmax (h) AUC0→120 (μg/mL · h) AUC0→∞ (μg/mL · h)
Fluconazole 29.60 ± 4.98 5.76 ± 0.75 2.50 ± 0.89 224.60 ± 28.25 241.40 ± 32.37
Declaration of Interest
▼
The authors report no conflicts of interest.
Song J-j et al. Determination of Fluconazole … Drug Res 2015; 65: 52–56
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