Development of a fast LC-MS/MS assay for the
determination of deferiprone in human plasma and
application to pharmacokinetics
Ta-Shu Songa,b, Yow-Wen Hsieha,c, Ching-Tien Pengc,d,
Cheng-Hsiung Liua, Tai-Lin Chene and Mann-Jen Houra*
* Correspondence to: M.-J. Hour, School of Pharmacy, ChinaMedical University, Taichung 404, Taiwan. E-mail: [email protected]
a School of Pharmacy, China Medical University, Taichung 404, Taiwan b Research department, Yung Shin Pharmaceutical Ind. Co. Ltd, Taiwan c China Medical University Hospital, Taichung 404, Taiwan
d Department of Biotechnology, Asia University, Taichung, Taiwan
e Institute of Biochemistry and Molecular Biology, National Yang-Ming University, Taipei, Taiwan Abbreviations used: EDTA, disodium dehydrogen ethylenediamine tetraacetate dihydrate.
ABSTRACT: A fast and accurate liquid chromatography/tandem mass spectrometric
(LC-MS/MS) assay was frst developed and validated for the determination of deferiprone in human plasma. The analytes were extracted with acetonitrile from only 50 mL aliquots of human plasma to achieve the protein precipitation. After extraction, chromatographic separation of analytes in human plasma was performed using a Synergi Fusion-RP 80A column at 30 _C. The mobile phase consisted of methanol and 0.2% formic acid containing
0.2mM EDTA (60:40, v/v). The fow rate of the mobile phase was 0.8 mL/min. The total run time for each sample analysis was 4 min. Detection was performed using
electrospray ionization in positive ion multiple reaction monitoring mode by monitoring the precursor-to-parent ion transitions m/z 140.1!53.1 for deferiprone and m/z 143.1!98.1 for internal standard. A linear range was established from 0.1 to 20mg/mL. The limit of detection was determined as 0.05mg/mL. The validated method was estimated for linearity, recovery, stability, precision and accuracy.
Intraday and interday precisions were 4.3–5.5 and 4.6–7.3%, respectively. The recovery of deferiprone was in the range of 80.1–86.8%. The method was successfully applied to a pharmacokinetic study of deferiprone in six thalassemia patients.
Keywords: LC/MS/MS; deferiprone; plasma samples; pharmacokinetics
Introduction
Iron chelators are needed to prevent damage to the heart, liver and endocrine glands from iron overload in patients with refractory anemia who receive regular blood transfusions. Deferiprone (1,2-dimethyl-3-hydroxypyridin-4-one, Fig. 1), also known as L1, CP20, Ferripox and Kelfer, is the frst oral iron chelator to be used clinically, mainly in thalassemia patients
(Kontoghiorghes, 2001). Although
desferrioxamine nowadays is the frst-line iron chelating drug for treating transfusional iron overload, recent data suggest that deferiprone may be superior to desferrioxamine at protecting heart from iron overload (Anderson et al., 2002; Peng et al., 2003). Many patients are successfully chelated at a
dose of 75mg/kg per day, but not more than 100mg/kg per day (Olivieri et al., 1995). Deferiprone belongs to the family of a-ketohydroxypyridines. Early studies on thalassemia patients showed that the drug is effective in increasing urine iron excretion, the amount being related to the total daily dose of the drug (Hoffbrand, 2005; Kontoghiorghes et al., 1987). Deferiprone is rapidly absorbed from gastrointestinal tract with peak plasma concentrations occurring between 45 and 60min after oral
dose ingestion. Food reduces the rate of absorption, but not the amount of drug absorbed (Matsui et al., 1991; Sweetman, 2002). It forms 1:3 iron-deferiprone complexes that are excreted in urine as well as free drug. More than 90% of the free drug is eliminated from plasma in most patients within 3–6 h of ingestion. The mean elimination half-life was 160 and 91min in several studies (Matsui et al., 1991; Sweetman,
2002; Al-Refaie et al., 1995). Deferiprone is metabolized to an inactive glucuronide metabolite and excreted in the urine.
Drug analysis makes an extensive impact on medical care. In order to optimize the dosage regimen and minimize the risk of side effects, monitoring the drug therapy, measuring the drug level accurately in plasma or other body fuid, is necessary. Previously studies have shown that the chromatography of the 3-hydorxypyridin-4-ones is diffcult on normal octadecylsilica columns as the analytes are often characterized by broad asymmetrical and sometimes multiple peaks (Epemelu et al., 1990). Several bioanalytical methods have been reported using HPLC with UV method for the determination of deferiprone in biological fuid (serum, plasma and urine) (Goddard and Kontoghiorghes, 1990; Dresow et al., 1995; Klein et al., 1991; Liu et al., 1999). Goddard and Kontoghiorghes (1990) and Dresow et al. (1995) reported for the simultaneous determination of deferiprone and its metabolite the same serum process with a high chromatographic run time of over 20 min per sample analysis (Goddard and Kontoghiorghes, 1990; Dresow et al., 1995). An improved extraction
procedure (2*5mL CH2Cl2 under Ph 7.4 liquid extraction) for deferiprone in human plasma is
given by Klein et al. (1991) with a limit of quantitation of 0.5 g/mL using 0.25mL plasma (Klein et al., 1991). Recently, Limenta et al. (2008) presented an HPLC-UV method for deferiporne and metabolite using 0.5mL human serum through ultrafltration. The limit of quantifcation for the analytes was 1 g/mL.
To meet the demands for the pharmacokinetic study, a method with high selectivity and specifcity, small volume samples and rapid turnaround time is highly desirable. Thus, the aim of the study was to optimize and validate a high-throughput LC-MS/MS method for
routine quantifcation of deferiprone in a small volume (50 L) of human plasma after a simple extraction step and a short run time of 4 min for chromatographic separation in support of clinical fndings. The validated method has been successfully applied to a pharmacokinetic study of commercial deferiprone tablets (L1, 80mg/kg) in six patients.
Experimental
Chemicals and reagents
Deferiprone (purity 98%) was purchased from Sigma-Aldrich (Germany). Piracetam (purity 100%) was kindly provided by Yung Shin Pharmaceutical Ind. Co. Ltd, Taiwan, and used as an internal standard (IS). HPLC-grade acetonitrile and methanol were purchased from Burdick & Jackson (SK Chemicals, Korea). grade formic acid was purchased from Merck (Darmstadt, Germany). Analytical-grade disodium dehydrogen ethylenediamine tetraacetate
dihydrate (EDTA) was from Hayashi Pure Chemical Ind. Ltd (Osaka, Japan). Deionized water was produced by a water purifcation system (Modulab ModuPure plus, Continental, Australia). Drug-free plasma was obtained from the local blood center, Taichung, Taiwan.
Instrumentation and chromatographic conditions
A vortex mixer (Scientifc Industries, USA) and analytic evaporation concentrator (Organomation Associates, USA) were used in the sample preparation. The HPLC system consisted of an Agilent 1100 series with an inline degasser, a binary pump, a column thermostat (Agilent Technologies, USA) and a CTC Analytics AG autosampler (PAL system, Switzerland). The separation was performed on a Synergi Fusion-RP 80A column (4 m, 150*4.6mm i.d.; Phenomenex, USA). The isocratic mobile phase, a mixture of methanol and 0.2% formic acid containing 0.2mM EDTA (60:40, v/v), was fltered through a 0.45m Millipore membrane flter and degassed prior to use. The trace amount of EDTA added is to prevent the interference caused by iron ions, which were released from the metal tubes. The mobile phase was delivered at a fow rate of 0.8mL/min with a split ratio of 1:9. The column oven temperature was maintained at 30 ℃.
The plasma concentration of deferiprone was quantifed by using LC-MS/MS spectrometry with a API 4000 Q-trap triple quadruple mass spectrometer (Applied Biosystems, USA) equipped with a turbo V electrospray ionization (ESI) source in the positive ion mode, heated to 500℃, and its ion spray voltage set to 5500 V. Quantitation was performed using multiple reaction monitoring mode to study parent→product ion transition of m/z 140.1→53.2 for
deferiprone and m/z 143.1→98.2 for piracetam (IS), respectively.The compound-dependent optimal MS parameters obtained were as follows: declustering potential (DP), 71 V, collision energy (CE), 50 V, and cell exit potential (CXP), 8 V for deferiprone; and DP, 31 V, CE, 13 V, and CXP, 10 V for piracetam. Nitrogen was used as curtain gas, 10 psig, and collision activation dissociation gas, medium. Automated data acquisition and analysis were performed using Analyst software (version 1.4).
control samples
The stock solution of deferiprone (1.0mg/mL) was prepared by dissolving in methanol. The IS (piracetam) was prepared as a stock solution (0.2 mg/mL) in methanol and was further diluted with acetonitrile to give a concentration of 20.0 g/mL. The stock solution of deferiprone was further diluted with 50% methanol (methanol:water, 1:1, v/v) to obtain working solutions at concentrations of 1.0, 2.0, 5.0, 20.0, 50.0, 100.0 and 200.0 g/mL. The calibration curve samples at concentrations of 0.1, 0.2, 0.5, 2.0, 5.0, 10.0 and 20.0 g/mL of deferiprone were prepared daily by serially
diluting a stock solution with drug-free plasma. The quality control (QC) samples were prepared in the same way at concentrations of 0.2 (low), 1.0 (medium) and 15 (high) g/mL of deferiprone. All plasma samples were stored at -80 ℃. Twenty fve microliters of IS (20.0 g/mL) was added to 0.05mL of calibration curve samples and quality samples. The further processing of both the calibration curve samples and the quality control samples was the same as described in the following section for preparation of the samples. All the stock solutions were stored at -10℃ and were brought to room temperature before use.
Sample preparation
To 50 L aliquot of plasma sample, which was placed into a 15mL clean glass test tube, 25 L of IS working standard (20g/mL) was added. The sample mixture was stirred for about 10 seconds using a vortex mixer. After the addition of 300 L of acetonitrile to the mixture, it was vortex-mixed for another 3 min, then centrifuged at 1350 g for 5 min. The resulting supernatant solution was transferred into another clean glass tube where it was evaporated to dryness
using an analytic evaporation concentrator under a stream of nitrogen at 45℃. The dry residue was reconstituted with 1.5mL of 50% methanol (methanol:water, 1:1, v/v), then vortex-mixed for 1min, from which 10 L aliquots were injected into the LC-MS/MS system, with a split ratio of 1:9.
Method validation
The method was validated in accordance with current acceptance criteria. Selectivity was evaluated by analyzing human plasmas from six different drug-free sources to investigate the potential
interferences at the retention times of deferiprone and IS. The acceptance criterion for experiment was that there were no signifcant interfering peaks at retention time of the analysis. Standard plasma samples at the lowest concentration of 0.1 g/mL were assayed as the lower limit of quantitation (LLOQ) samples. Relative errors of the six replicates of LLOQ samples analyzed to validate the method were required to be within 20% of the target value.
Calibration curves were obtained by analyzing standard plasma samples at seven concentrations of 0.1, 0.2, 0.5, 2.0, 5.0, 10.0 and 20.0 g/mL. The linear regression equation was y = a + bx with a weighting factor of 1/x, in which y is the peak area ratio of deferiprone to IS and x is the concentration of the deferiprone.
The within- and between-run precisions were expressed as relative coeffcient variation (CV) and the accuracy was expressed as relative error (RE). Within-run assay on each day consisted of a set of calibrations standards and six batches of QC samples at three concentrations (0.2, 1 and 15 g/mL, n
= 6). The between-run assay was evaluated by analysis of six batches of QC samples on six different days. The accuracy and precision were required to be within ±15% of the nominal concentration and <15% CV for all three concentration levels of QC samples.
The recovery was measured by comparing the response of deferiprone from spiked plasma samples with the response of deferiprone from a standard solution at the same analysis concentration prepared in methanol–water (1:1, v/v) and processed in the same manner as for the plasma sample. The recovery was evaluated for each QC level. The matrix effects were assessed by comparison of the mean peak areas of the analytes at three QC concentrations spiked into postextraction plasma extracts originating from six different humans with the mean peak areas for neat solutions of the analytes in 50% methanol.
The stability procedures were evaluated by the stability of the deferiprone in the human plasma under distinct timing and temperature conditions. In this study, the freeze–thaw stability through three cycles, autosampler stability and long-term stability were determined out. For freeze–thaw stability, QC samples were subjected to three cycles from -20 ℃ to room temperature. The autosampler stability was assessed by placing processed QC samples in an autosampler at 10℃ for 24 h, and long-term stability was evaluated by freezing QC samples at -20 ℃ for a month. The samples were considered to be stable if assay values were within the acceptance limits of accuracy (±15% RE) and precision (±15% CV).
Pilot pharmacokinetic study
The pharmacokinetic study was approved by the Institutional Review Board (IRB) of China Medical University Hospital (Taichung, Taiwan). All volunteers signed an informed consent prior to participating in this study. Six thalassemic patients, one female and fve males, with mean age of 20.7±3.7 years (15–26 years), mean height of 163.8±4.9 cm (156.3–169.8 cm) and mean body weight of 53.5±5.1 kg (49–60 kg) were selected for this study. They had not received chelation therapy during the preceding 3 days or the day after the study.
All subjects underwent overnight fasting before the treatment. Each patient received orally a single dose of L1 tablet (80mg/kg) with 240mL of water. They were allowed only water for the next 4 h. Blood samples were collected at baseline prior to the treatment and at 0.25, 0.5, 0.75, 1.0, 1.5, 2, 3, 4, 5, 6, 7 and 10h after the drug administration. Plasma was immediately separated and kept at -20 ℃ before analysis. The pharmacokinetic parameters were calculated from the plasma drug concentration using the software WinNonlin (version 2.0, Pharmasight, USA).
Results and discussion
Mass spectrometry
The Q1 spectra of deferiprone and piracetam (IS) were scanned by infusion fow with a positive ion interface. The protonated precursor ions [M+ H]+were produced at m/z 140.1
and 143.1 for deferiprone
98.1, respectively. The product ion mass spectra of deferiprone (A) and piracetam (B) are shown in Fig. 2.
Method validation
Selectivity and matrix effect. The selectivity and specifcity of the method were
evaluated by comparing the chromatograms of six different sources of blank human plasma with peak response of deferiprone and piracetam (IS) at LLOQ (0.1 g/mL). There were no signifcant interfering peaks across the retention window of deferiprone and IS. The typical chromtograms of blank plasma and spiked plasma with 0.1 mg/mL of deferiprone are shown in Fig. 3. As shown, the retention times for deferiprone and IS were about 1.78 and 2.26min, respectively. The total LC-MS/MS analysis time was about 4.0min per sample. The matrix effect of deferiprone at 0.2, 1.0 and 15.0 g/mL was 91.0±0.06, 98.7±0.01 and 99.4±0.03 %, respectively. The matrix effect of the IS was evaluated in a similar way and the result was 103.2% (CV<10%). The results indicated that no endogenous substances interfere with the ionization of the deferiprone and a slight ionization enhancement occurs for the IS. This data suggest that the matrix effects for deferiprone and piracetam have little effect on determination of deferiprone in human plasma samples.
Limit of quantitation.
The limit of detection was assessed with a plasma sample spiked with deferiprone to the fnal concentration of 0.05 g/mL. The limit of detection was determined as 0.05 mg/mL, which provided the signal-to-noise ratio of approximately 3:1. The LLOQ for deferiprone in plasma was 0.1 g/mL, which was suffcient for pharmacokinetic study of deferiprone in plasma samples. At the concentration level,
the precision and accuracy at the LLOQ were 5.9 and 2.5%, respectively.
Linearity and calibration curves. The linearity of each calibration curve was obtained
following regression of the peak ratio vs plasma concentration, and was ftted (weighting factor of 1/x, where x is the nominal concentration) over the concentration range of 0.1–20 g/mL. The calibration curve exhibited good linearity (r>0.999) and
showed good back-calculated precision and accuracy (Table 1). The typical regression equation of calibration curve was y=0.759x + 0.00616 (r = 0.9998, where y is peak area ratio and x is plasma concentration). As showed in Table 1, the back-calculated
concentrations were within the limits of acceptance.
The impact of EDTA in mobile phase. In order to prevent the formation of iron
complexes of the deferiprone with ferric ion present in the samples and the metal tubes of the HPLC system, a trace amount of EDTA (0.2 mM) was included in the mobile phase. When the mobile phase containing EDTA was applied, the peak intensity of deferiprone was not affected and the peak intensity of piracetam was decreased. This demonstrated that the impact of EDTA in mobile phase was minor on deferiprone. The impact of EDTA on the
IS was more obvious and therefore the concentration of 20 g/mL IS was suitable for analysis. However, the mass instrument may have been contaminated by EDTA. To prevent the interference in mass caused by EDTA, it is necessary to split the mobile phase after the column and conduct periodic frst-level (orifce) maintenance of mass during analysis. The amount of EDTA in mobile phase should be minimized for the special needs of LC MS/MS.
Precision and accuracy. Precision and accuracy studied were satisfactory at QC
concentrations. The QC samples of deferiprone for within- and between-run data are shown in Table 2. The CVs within- and between-run were 4.3–5.5 and 4.6–7.3%, respectively. Relative errors were _5.2–1.6 and _5.3–0.3%, respectively. Thus, the results obtained were reproducible and satisfed the criteria for acceptance of
precision and accuracy.
Recovery. The mean recovery of deferiprone determined at three different concentrations
(0.2, 1 and 15 g/mL) were 86.9, 82.8 and 80.1% (n = 3), respectively. The mean recovery of IS (piracetam) was 95.0% (n = 6). The results showed that the recoveries of deferiprone and IS were consistent and reproducible. Thus the analytical procedure was satisfactory for the assay of samples.
Stability. Triplicate QC samples at nominal concentrations of 0.2, 1 and 15 g/mL were subjected to three freeze–thaw cycles with each cycle lasting at least 12 h of freeze. All the samples were analyzed on the same day and the results were compared with the
calculated mean concentrations listed in Table 3. After three freeze–thaw cycles, the observed mean concentration deviated less than 10% at the three concentrations. The result indicated that repeated freeze–thaw cycles did not affect the sample integrity of deferiprone in human plasma. Leaving the QC samples in the autosampler at 10℃ for 24 h also did not affect the concentrations and nature of plasma (Table 3).
The long-term stability was evaluated by freezing QC samples (0.2, 1 and 15 g/mL) at -20℃ for up to 56 days. The differences from freshly prepared samples were about 5.0, -0.1 and 5.3%, respectively. The results of stability tests were well within the acceptable limits. Furthermore, the results revealed that no signifcant degradation occurred during the plasma samples treatment. The data for different stability tests in plasma and values for the precision and accuracy expressed as CV% and RE% are listed in Table 3.
According to the results described above, the developed LC-MS/MS method presented for the analysis of deferiprone in plasma samples demonstrated good precision and accuracy.
Application to a pharmacokinetic study
The developed method in this paper was successfully used for a pilot pharmacokinetic study in which plasma concentration of deferiprone up to 10 h after oral administration a single dose of L1 tablet (80 mg/kg) in six patient volunteers at China Medical University Hospital. The mean plasma
parameters are summarized in Table 4. The mean terminal half-life (T1/2) was 3.5±1.6 h; the
mean maximum concentration (Cmax) was 12.8±4.0 mg/mL; the mean time to maximum
concentration (Tmax) was 1.8±0.9 h; and the mean area under the plasma drug
concentration– time curve (AUC0–t) and (AUC0–1) were 44.9±11.1 and 52.5±14.3 mgh/mL.
Conclusions
In this study we report for the frst time the development and validation of a simple, fast and accurate highperformance liquid chromatography–tandem mass spectrometry (LC-MS/MS) method for the determination of deferiprone in human plasma. This method showed good linearity, specifcity, precision and accuracy over the range of therapeutic plasma concentrations. Thereby, patient therapy could be closely monitored through a validated method.
This method demonstrated some advantages as follows:
(1) One of the major advantages is that only 50 L of plasma is needed for analysis and the amount is more convenient for collecting the blood samples and making the impact on patients as small as possible.
(2) The protein precipitation extraction using acetonitrile is faster and more economical in this study.
(3) The LC-MS/MS assay requires a run time of only 4min to fnish the work in the study. From the illustration above, an LC-MS/MS assay for deferiprone in human plasma was developed. This method did not require complicated extraction and provided a fast
analysis. Validation results indicated that the method is specifc, reproducible and reliable. We suggest the method is suitable for pharmacokinetic study of deferiprone in human subjects.
Supporting information
Supporting information can be found in the online version of this article.
Acknowledgments
The authors would like to thank Dr Fang-Chen Lee, President, Yung Shin Pharm. Ind. Co. Ltd, Taiwan, for supporting this work. We also gratefully acknowledge the excellent manuscript suggestion provided by Dr Yi-Hui Lin.
References
Al-Refaie FN, Sheppard LN, Nortey P, Wonke B and Hoffbrand AV. Pharmacokinetcsof the oral chelator deferiprone (L1) in patients with iron overload. British Journal of Haematology 1995; 89: 403–408. Anderson LA, Wonke B, Prescott E, Holden S, Walker JM and Pennell DJ. Comparison of effects of oral deferiprone and subcutaneous desferrioxamine on myocardial iron concentrations and ventricular
function in beta-thalassaemia. The Lancet 2002; 36: 516–520.
Dresow B, Fischer R, Janka GE and Gabbe EE. HPLC-based measurement of the chelator 1,2-dimethyl-3-hydroxy-pyrid-4-one (L1) and its iron complex for pharmacokinetic studies in humans. Frensenius’ Journal of Analytical Chemistry 1995; 352: 562–564.
Epemelu RO, Singh S, Hider RC and Damani LA. Chromatography of 3-hydroxypyridin-4-ones; noval orally active iron chelators. Journal of Chromatography. A 1990; 519: 171–178.
Goddard JG and Kontoghiorghes GJ. Development of an HPLC method for measuring orally
administered 1-substituted 2-alkyl-3-hydroxypyrid- 4-one iron chelators in biological fuids. Clinical Chemistry
1990; 36: 5–8.
Hoffbrand AV. Deferiprone therapy for transfusional iron overload. Best Practice & Research. Clinical Haematology 2005; 18: 299–317.
Klein J, Damani D, Chung D, Epemoulu O, Olivieri N and Koren G. A high performance liquid
chromatographic method for the measurement of the iron chelator 1,2-dimethyl-3-hydroxypyridin-4-one in human
plasma. Therapeutic Drug Monitoring 1991; 13: 51–54.
Kontoghiorghes GJ. Clinical use, therapeutic aspects and future potential of deferiprone in thalassemia and other conditions of iron and other metal toxicity. Drugs of Today 2001; 37: 23–35.
Kontoghiorghes GJ, AldouriMA, Hoffbrand AV, Barr J, Wonke B, Kourouclaris T and Sheppard L, Effective chelation of iron in beta thalassemia with the oral chelator 1,2-dimethyl-3-hydroxypyri-4-one. British Medical Journal
1987; 295: 1509–1512.
Limenta LMG, Jirasomprasert T, Tankanilert J, Svasti S, Wilairat P, Chantharaksri U, Fucharoen S and Morales NP. UGT1A6 genotype-related pharmacokinetics of deferiprone (L1) in healthy volunteers. British Journal of Clinical Pharmacology 2008; 65: 908–916.
Liu DY, Lui ZD, Lu SL and Hider RC. Liquid extraction and ion-pair HPLC for determination of hydrophilic 3-hydroxypyridin-4-one iron chelators. Journal of Pharmaceutical and Biomedical Analysis 1999; 21: 759–765.
Matsui D, Klein J, Hermann C, Grunau V, McClelland R, Chung D, St-Louise P, Olivieri N and Koren G. Relationship between the pharmacokinetics of the new oral iron chelator, 1,2-dimethyl-3-hydroxypyrid-4-one in patients with thalassemia. Clinical Pharmacology and Therapeutics 1991; 50: 294–298. Olivieri NF, Brittenham GM, Matsui D, Berkovitch M, Blandis LM, Cameron RG, McClelland RA, Liu PP, Templeton DM and Koren G. Iron-chelation therapy with oral deferiprone in patients with thalassemia major. The New England Journal of Medicine 1995; 332: 918–922.
Peng CT, Chow KC, Chen JH, Chiang YP, Lin TY and Tsai CH. Safety monitoring of cardiac and hepatic systems in beta-thalassemia patients with chelating treatment in Taiwan. European Journal of Haematology 2003; 70: 392–397.
