Therapeutic drug monitoring of Mitotane: analytical assay and patient follow-up
Yoann Cazaubon, Department of Pharmacology, E.A.3801, Centre Hospitalier Universitaire de Reims, Reims, France. E-mail: ycazaubon@chu-reims.fr
Helene Guillemin, Department of Pharmacology, E.A.3801, Centre Hospitalier Universitaire de Reims, Reims, France. E-mail: hguillemin@chu-reims.fr
Damien Vautier, Department of Pharmacology, E.A.3801, Centre Hospitalier Universitaire de Reims, Reims, France. E-mail: dvautier@chu-reims.fr
Olivier Oget, Department of Pharmacology, E.A.3801, Centre Hospitalier Universitaire de Reims, Reims, France. E-mail: ooget@chu-reims.fr
Hervé Millart, Department of Pharmacology, E.A.3801, Centre Hospitalier Universitaire de Reims, Reims, France. E-mail: hmillart@chu-reims.fr
ACGPDIPOLA
Claire GOZALO, Department of Pharmacology, E.A.3801, Centre Hospitalier Universitaire de Reims, Reims, France. E-mail: cgozalo@chu-reims.fr
Zoubir Djerada, Department of Pharmacology, E.A.3801, Centre Hospitalier Universitaire de Reims, Reims, France. E-mail: zdjerada@chu-reims.fr
*Corresponding Author: Catherine Feliu, Department of Pharmacology, E.A.3801, Centre Hospitalier Universitaire de Reims, 51, rue Cognacq-Jay, Reims, France, 51092. E-mail: catherine.feliu@univ-reims.fr
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/bmc.3993
Abstract
☒
Background: Adrenocortical carcinoma (ACC) is an aggressive malignancy of the adrenal gland. Mitotane (o,p’-DDD) is the most effective chemotherapy for ACC. According to literature, mitotane plasma trough concentrations within 14-20 mg.L1 are correlated with a higher response rate with acceptable toxicity. Therapeutic drug monitoring (TDM) of mitotane is therefore recommended. The aim of this study was to propose a robust, and simple method for mitotane quantification in plasma. The validation procedures were based on international guidelines.
Methods: Sample preparation consisted in a single protein precipitation with methanol using 100 ML of plasma. The supernatant was submitted to liquid chromatography coupled with ultra-violet detection at 230 nm.
☒ Results: Mitotane retention time was 7.1 minutes. Limit of detection was 0.1 mg.L-1 and limit of quantification was 0.78 mg.L-1. The assay demonstrated a linear range of 0.78 to 25 mg.L-1 with correlation coefficients (12) at 0.999. Inter- and intra-assay precision were less than 4.85 %. Evaluation of accuracy showed a deviation less than 13.69 % from target concentration at each quality control level.
Conclusion:
This method proved easy and rapid to perform mitotane TDM and required a small volume of sample. It was successfully applied to routine TDM in our laboratory.
Keywords
· Mitotane, HPLC-UV, therapeutic drug monitoring
List of abbreviations:
☒ ACC : Adrenocortical carcinoma
· HPLC-UV high-performance liquid chromatography coupled with ultra-violet
☒ detection
· o,p’-DDA : 1,1-(o,p’-dichlorodiphenyl) acetic acid
· o,p’-DDD: 1,1-(o,p’-dichlorodiphenyl)-2,2-dichloroethane
· o,p’-DDE : 1,1-(o,p’-dichlorodiphenyl)-2,2 dichloroethene
. QC quality control
Introduction
☒
Adrenocortical carcinoma (ACC) is an uncommon aggressive malignancy of the adrenal gland that usually results in a higher than normal hormone production (PDQ Adult Treatment Editorial Board, 2002). Clinical symptoms depend on the types of hormones produced in large amount i.e. cortisol, aldosterone, testosterone and / or oestrogens (PDQ Adult Treatment Editorial Board, 2002). Radical surgery is the treatment of choice (PDQ Adult Treatment Editorial Board, 2002). Unfortunately, even after successful excision, local or metastatic recurrence is frequent. Mitotane (1,1-(o,p’-dichlorodiphenyl)-2,2-dichloroethane, o,p’-DDD) is the most effective drug therapy for ACC (Paci et al., 2014; PDQ Adult Treatment Editorial Board, 2002), although an important limitation for its use is its toxicity. Hepatic disorder, leukopenia, neurologic symptoms and gastrointestinal disturbances are the most frequent side-effects described in literature (Daffara et al., 2008). They occur in all patients but can be controlled with palliative treatment and adjustment of hormone replacement therapy. Therapeutic drug monitoring of mitotane is described in many studies ☒ as a useful tool to manage efficacy (Van Slooten et al., 1984; Baudin et al., 2001) and to
reduced toxicity (Van Slooten et al., 1984; Baudin et al., 2001). According to these studies, mitotane plasma trough concentrations within 14-20 mg.L- are correlated with a higher response rate and longer survival with acceptable toxicity. Mitotane plasma concentrations higher than 20 mg.L- are described as a risk factor of neurotoxicity and offer no further benefit in terms of efficiency (Van Slooten et al., 1984; Baudin et al., 2001). Neurotoxicity seems to be related to the cumulative exposure of mitotane.
After oral administration, mitotane undergoes a biotransformation into o,p’-DDA as major metabolite and o,p’-DDE (minor metabolite) (Kasperlik-Zaluska et al., 2005).
Mitotane is a lipophilic xenobiotic and thus accumulates in fatty tissues. At the end of the treatment, it is released from fatty tissues with a long terminal half-life ranging from 17 to 159 days (« European Medicines Agency - Find medicine - Lysodren »). Some studies evaluated the therapeutic drug monitoring of mitotane and its metabolites as predictive factors of tumor response with discordant results (Hermsen et al., 2011; Kasperlik-Zaluska et al., 2005). Kasperlik-Zaluska et al. found that a higher plasma concentration of o,p’-DDE and a higher o,p’-DDE/o,p’-DDD ratio seem to be a good prognostic factor during prolonged mitotane therapy (Kasperlik-Zaluska et al., 2005). On the contrary, Hermsen et al., found no significant relationship between tumour response and o,p’-DDE plasma concentration but they suggested an additional benefit of combined o,p’-DDD and o,p’-DDA monitoring in ACC treatment (Hermsen et al., 2011). Several years later, Hescot et al. reported a lack of antitumor effects for o,p’DDA which excluded its role as an active metabolite of mitotane for adrenocortical carcinoma treatment (Hescot et al., 2014). Thereby, the only predictive factor of tumor response is mitotane (o,p’-DDD) plasma concentrations. ☒ ☒
Several dosing regimens have been proposed to achieve a plasma concentration between 14 and 20 mg.L”. Low dosing regimens begin with 3 g per day for 3 to 4 months then continue with 1 to 2 g per day and allow to achieve the therapeutic range with manageable side effects
in 3 to 5 months (« European Medicines Agency - Find medicine - Lysodren >>; Terzolo et al., 2000). High dosing regimens consisting in progressively attaining 4 to 9 g per day within 2 weeks and maintaining this dose at least 6 weeks allow to reach the therapeutic range earlier, sometimes within 4 weeks, with an acceptable tolerance (Faggiano et al., 2006, Mauclère- Denost et al., 2012). The delay to reach the steady state and the therapeutic range is due to mitotane’s very long elimination half-life. Therefore, it is recommended to perform mitotane plasma monitoring regularly at treatment initiation and after each dose adjustment until
optimal concentration is achieved. A control every 15 days seems to be adapted. But more frequent mitotane monitoring, for example once a week, is recommended when a high dosing regimen is used. In case of treatment interruption, regular monitoring every two months is also required to control the concentration decrease (« European Medicines Agency - Find medicine - Lysodren »).
To date, several methods have been proposed for mitotane therapeutic drug monitoring (Andersen et al., 1999; De Francia et al., 2006; Garg et al., 2011; Mornar et al., 2012). Methods based on gas chromatography coupled with mass spectrometry require an expensive equipment and use toxic solvents for extraction (Benecke et al., 1987; Inouye et al., 1987). Methods based on liquid chromatography coupled with ultra-violet detection have complex procedures for sample pre-treatment involving liquid/liquid extraction, or liquid extraction coupled with solid phase extraction (Andersen et al., 1999; De Francia et al., 2006; Garg et al., 2011; Mornar et al., 2012).
The aim of this study was to validate a simple, fast, and robust method for the analysis of mitotane in human plasma using high-performance liquid chromatography (HPLC) with ultra-violet (UV) detection. This method requires only 100 ul of plasma sample. Sample pre- treatment consist only in a deproteinisation step. This method was successfully implemented
in our routine laboratory, as demonstrated in three examples.
2. Materials and methods
2.1. Chemicals
Mitotane, o,p’-DDA and o,p’-DDE were purchased from Sigma Aldrich (Misssouri, United States). Acetonitrile, methanol, tetramethylammonium chloride, orthophosphoric acid and potassium hydrogen phosphate were purchased from VWR (Pennsylvania, United States). Potassium dihydrogen phosphate was purchased from Merck (Darmstadt, Germany). Ultrapure water was obtained from Biosolve (Dieuze, France). Plasma from healthy donors was purchased from the Jacques Boy Institute of Biotechnology (Reims, France). 4
2.2. Chromatographic conditions
Analyses were realized on an Alliance high performance liquid chromatographic system (Waters Corp, Milford, MA, USA) coupled with ultra-violet detector (Waters Corp, Milford, MA, USA).
Chromatographic separation was achieved with an Uptisphere 5 M ODB pre-column from Interchim (reference: CH979520, Montlucon, France) and a LiCHROSPHER 100 RP-8 (5 um, 250 x 4 mm) column from Merck (Darmstadt, Germany) maintained at 25 ℃. The mobile phase consisted of a phosphate buffer pH 3 (mobile phase A) and acetonitrile (mobile phase B) at the ratio of 25:75 (v/v). Mobile phase A was prepared as follows: tetramethylammonium chloride (TMA) 0.02 M, potassium hydrogen phosphate 0.01M in water at pH 3 (orthophosphoric acid). The use of TMA eliminated the undesirable secondary interactions due to free silanols on the bonded silica column, providing effective and reproducible separation. Isocratic flow rate was set to 1.2 mL.min. Ultra-violet detection was performed at 230 nm with diode array detector. The run time was 8 minutes.
2.3. Preparation of stock solutions, calibration standards and quality control samples
Stock solution was prepared in methanol to obtain a final concentration of 1 g.L-1. This solution was stocked at -5 +/-3℃. Five calibration solutions were prepared from this working solution by serial dilution (1/2) with methanol. Twenty-five microliters of working solution, each calibration solution or methanol were then spiked in 100 uL plasma to achieve concentrations of 25, 12.5, 6.25, 3.125, 1.56, 0.78 and 0 mg.L-1 for the seven calibrators. A different stock solution was used to prepare high, medium and low-level quality controls, QCH (20 mg.L-1), QCM (11 mg.L-1) and QCL (5 mg.L-1). Moreover, six levels of quality control (QC1, QC2, QC3, QC4, QC5 and QC6) were collected from an interlaboratory ☒ ☒
comparison scheme (number of participating laboratories = 5).
2.4. Sample processing
Precipitation was performed by adding a final volume of 250uL of methanol in a 100uL plasma volume (patient, QC or calibrator). The samples were then vortex-mixed during 30 seconds and centrifuged at 10.000 g for 4 minutes. Fifty microliters of supernatant were finally injected into the chromatographic column.
2.5. Validation procedure
☒ Method validation was realized in accordance to international recommendations (« Bioanalytical Method Validation -FDA », « European Medicines Agency - », Viswanathan et al., 2007). ☒
☒
2.5.1. Calibration curve and limits of quantitation
The limit of detection (LOD) was defined at mean + 3 standard deviations of the response from 10 blank samples.
The lower limit of quantitation (LLOQ) was defined as the lowest calibrator that would, in 6 replicates, with accuracy between 80 % and 120 % and a precision within 20 %. The upper limit of quantitation (ULOQ) was defined as the highest calibrator that would, in 6 replicates, with accuracy between 80 % and 120 % and a precision within 20 %.
Accuracy of the quality controls, analysis of the distribution mode of deviations from prediction of the calibration (Gaussian distribution verified by the D’Agostino Pearson test), and accuracy of standards within ±15% (± 20% at LLOQ) of nominal concentration were criteria to determine the regression mode.
2.5.2. Precision and accuracy
Precision and accuracy were evaluated with QCL (5 mg.L-1), QCM (11 mg.L-1) and QCH (20 mg.L-1). Within-run accuracy and precision were assessed by performing a calibration curve with six replicates of each QC in a single run. Between-run accuracy and precision were evaluated by performing a calibration curve with the three levels of controls on at least 6 occasions in 6 months. Accuracy was also evaluated in an interlaboratory (n=5) comparison scheme, with QC1, QC2, QC3, QC4, QC5 and QC6.
The limits defined by international recommendations for accuracy and precision (expressed as the relative standard deviation) are ±15% and ≤15%, respectively.
2.5.3. Selectivity
☒ To investigate interferences, six plasma samples from different donors were analysed individually as blanks. As recommended, absence of interfering components was characterized by blank responses lower than 20 % of the LLOQ for the analyte. In addition, potential interferences with mitotane metabolites were searched for by spiking samples with o,p’-DDA and o,p’-DDE at 20 mg.L-1. Finally, potential interferences were also investigated by spiking plasma with common co-medications of mitotane (synthetic corticosteroids like hydrocortisone, fludrocortisone, prednisone, prednisolone, dexamethasone and antiemetics drugs like ondansetron, haloperidol and metoclopramide) at their therapeutic concentration.
2.5.6. Stability
Stability of mitotane in plasma is largely described in the literature (Garg et al., 2011; Inouye et al., 1987; Mornar et al., 2012). ☒ Stability of the standard solution was evaluated by comparing a one-month solution to a freshly-prepared one.
The stability of the laboratory-made internal controls was tested over 1 year by comparing a batch of controls stored at -20℃ during 1 year and a batch of freshly prepared ones.
Post-preparative stability was evaluated by keeping processed samples placed in their glass vial for 3 h in autosampler (+ 18 ℃).
2.5.7. Carry over effects
Carry over was evaluated by analysis of blank samples injected right after the highest calibrator (25 mg.L-1) (n=6). The absence of carry-over effects is characterized by a mean signal for the blank samples lower than 20% of the signal measured at the LLOQ.
2.6. Therapeutic drug monitoring
The major interest of TDM of mitotane is illustrated by 3 typical situations: start of treatment, steady-state and cessation of treatment.
Ethics committee approval and patient consent are not compulsory in France in order to use retrospectively therapeutic drug monitoring data, so no informed consent had to be collected.
2.7 Statistical analysis
Statistical analyses were performed with Prism 4.00 (GraphPad Software. San Diego. CA).
3. Results
3.1. Linearity, precision and accuracy
Chromatography was achieved within 8 minutes with mitotane retention time around 7.1 minutes (figure 1).
Linear regression (Y = aX; mean ± sd of a: 31100 ± 1500, n = 6) without weighting fulfilled all predefined criteria. Over the considered concentration range, regression coefficient (r2) of the calibration curves were greater than 0.999 (n = 6) with peak purities superior than 98% by spectral examination. Back calculated calibrators concentrations were within ± 15 % (± 20 % at LLOQ) of nominal concentration (figure 1). ☒
The precision and accuracy at the LLOQ and ULOQ (table 1) for mitotane were within recommendations: 4.62 % and 114.74 % respectively at 0.78 mg.L-1; 4.79% and 93.75% respectively at 20mg.L. Limit of detection (LOD) was established at 0.1 mg.L-1. The relative standard deviations of quality controls (table 1) were between 0.83 and 4.85 % for
both intra- and inter-assay precision and were within acceptance criteria. Evaluation of accuracy of quality controls showed a relative standard deviation inferior to ± 15 % from target concentration at each tested level (100.83%-113.69%) (table 1). External quality controls showed a good accuracy (table 2).
3.2. Specificity and selectivity
Analysis of 6 different blank samples did not show any interference (< 5% of LLOQ for mitotane) at the retention time.
Mitotane retention time was around 7.1 minutes. Mitotane metabolites did not show any interference on mitotane quantification as retention times were 3.4, 7.2 and 9.3 minutes for o,p’-DDA, mitotane and o,p’-DDE respectively (figure 2). Drugs commonly coadministered with mitotane did not interfere with mitotane quantification.
3.3. Stability
Mitotane was found to be stable in processed samples (all standards) stored for 9 h in the autosampler. Mitotane stock solution and quality controls were found stable for 6 months at - 20℃.
3.4. Carry over effects
The absence of carry-over effects was demonstrated as the injection of blank samples after the highest calibrator showed no peak.
3.5 Example of Therapeutic drug monitoring
☒ This method was implemented in our laboratory for routine therapeutic drug monitoring, as shown in the three following examples (figure 3).
The first example describes the initiation phase of the treatment. A 25- year-old woman was treated by mitotane with a dosing regimen of 2 g twice a day. Regular therapeutic drug monitoring allowed to control the increase of mitotane concentration. Steady state concentration was achieved approximatively at 7 weeks (figure 3A). Steady-state was above 20 mg.L-1: this indicated the need for dose reduction to avoid toxicity.
The second example describes mitotane monitoring after a dose change (figure 3B). A 56- year-old man had been treated with mitotane 1 g twice a day for 4 years, with a steady state concentration between 16 and 18 mg.L”. Dose was reduced to 500 mg twice a day. Five months after the dose change, mitotane concentration fell under 14 mg.L-1. To avoid the risk of inefficacy due to its low concentration, the dose had to be increased again. This example shows the delay between dose change and mitotane concentration decrease and strengthens the benefit of therapeutic drug monitoring.
The last case describes the slow decrease of mitotane concentrations when the treatment is stopped. A 60-year-old woman had been treated by 1 g three times a day when the treatment ended. In this case, mitotane elimination followed one phase monoexponential decay (figure 3C) with an estimated half-life of 6 months. For this patient, mitotane was still detectable 2 years after the last dose. This example highlights the very long half-life of mitotane. Monitoring of its total elimination for months or years after the last dose is essential, because medical interactions can still be observed, the risk of teratogenicity persists …
To date, compiling all the data we have for patient who have benefited from therapeutic drug monitoring, the mean ± standard deviation [min-max] of trough mitotane plasma concentration 12 hours after administration was 11.6 ± 5.1 mg.L-1 (0.7-22.2 mg.L”), n =75) for a median (interquartile range) administered dose of 2.5 g/day (2.4-3.0 g/day). Two hours after administration, the plasma mitotane value was 16.46 ± 4.344 mg.L-1 (1.9-22.8 mg.L-1, n = 21) for a median (interquartile range) administered dose of 2.6 g/day (2.1-3.1 g/day).
4. Discussion
Various procedures have previously been described in the literature for the quantification of mitotane (Table 3). The first procedures for mitotane quantification were developed with gas chromatography after heptane or benzene extraction (Benecke et al., 1987; Inouye et al., ☒ 1987). More recently, quantification of mitotane with liquid chromatography coupled with ultra-violet detection has been proposed (Andersen et al., 1999; De Francia et al., 2006; Garg et al., 2011; Mornar et al., 2012). In addition to mitotane, all this techniques allowed quantification of its metabolites. We chose to focus on mitotane as no correlation has clearly been established to date between the metabolites’ s concentrations and the treatment’s efficacy (Hermsen et al., 2011; Hescot et al., 2014).
Compared to others method, our method needed smaller volume of sample (100 uL) than Garg et al. (200 uL) and Mornar et al. (500uL). Concerning sample preparation, we developed a simple precipitation with methanol. Mornar et al. proposed a liquid extraction followed by solid phase extraction. Gard et al. proposed precipitation with cold ethanol followed with addition of Potassium dihydrogen phosphate. As in our assay, Andersen et al. proposed a single precipitation of plasma proteins and used no internal standard. They used acetone as precipitating agent. Acetone is more volatile than methanol with a vapour pressure at 24.7 kPa for acetone, and 12.3 kPa for methanol. To improve robustness of the method without using an internal standard, methanol seemed to us more appropriate than acetone. Run times analysis was 8 minutes for our method, 10 minutes for Mornar et al. and 21 minutes for Garg et al. ☒
These three methods have assessed precision and accuracy. Mornar et al. have evaluated precision with one concentration at 20 mg/L (n = 18) and accuracy with three concentrations (1, 10 and 50 mg/L, n=3). Garg et al. have evaluated both precision and accuracy with two
concentrations (2.5 and 20 mg/L, n= 3). In our method we evaluated precision and accuracy with 5 concentrations included LLOQ and ULOQ (0.78, 5, 10, 20 and 25 mg/L, n =6). For all these three methods and as our methods, intra-assay and inter-assay variation and accuracy were according the guidelines recommendations (« Bioanalytical Method Validation -FDA », « European Medicines Agency - », Viswanathan et al., 2007): relative standard deviation less than 2.38% and accuracy less than 109.60 for Mornar et al. < 4.10 %, < 101.4 for Garg et al. and < 6.32%, 114.74 for our method. Proficiency test between laboratories were only reported in our work.
Conclusion
In this work, we describe the validation of a rapid, sensitive and accurate HPLC-UV method to quantify mitotane after a simple plasma deproteinization, according to recommandations of FDA and EMA (« Bioanalytical Method Validation - FDA», s. d., « European Medicines Agency - », s. d .; Viswanathan et al., 2007). The assay requires small volumes of biological sample and is not expensive regarding sample preparation. This assay is now successfully applied to routine therapeutic drug monitoring of mitotane in our laboratory.
Conflict of interest
None declared.
Acknowledgements:
This work was supported by Reims University Hospital, France.
Bibliography:
Andersen A, Kasperlik-Zaluska AA and Warren DJ. Determination of mitotane (o,p-DDD)
☒
and its metabolites o,p-DDA and o,p-DDE in plasma by high-performance liquid chromatography. Therapeutic Drug Monitoring 1999; 21(3): 355-9.
Baudin E, Pellegriti G, Bonnay M, Penfornis A, Laplanche A, Vassal G and Schlumberger,
M. Impact of monitoring plasma 1,1-dichlorodiphenildichloroethane (o,p’DDD)
☒ levels on the treatment of patients with adrenocortical carcinoma. Cancer, 2001; ☒ 92(6): 1385-92.
Benecke R, Vetter B and De Zeeuw RA. Rapid micromethod for the analysis of mitotane and its metabolite in plasma by gas chromatography with electron-capture detection. Journal of Chromatography, 1987; 417(2): 287-94.
☒ Bioanalytical Method Validation - FDA.
☒ http://www.fda.gov/downloads/drugs/guidancecomplianceregulatoryinformation/guid ances/ucm368107.pdf
Daffara F, De Francia S, Reimondo G, Zaggia B, Aroasio E, Porpiglia F, Volante M, Termine
☒ A, Di Carlo F, Dogliotti L, Angeli A, Berruti A and Terzolo M. Prospective
☒ evaluation of mitotane toxicity in adrenocortical cancer patients treated adjuvantly. Endocrine-Related Cancer, 2008; 15(4): 1043-3.
☒ https://doi.org/10.1677/ERC-08-0103
De Francia S, Pirro E, Zappia F, De Martino F, Sprio AE, Daffara F, Terzolo M, Berruti A , ☒ Di Carlo F and Ghezzo F. A new simple HPLC method for measuring mitotane and its two principal metabolites Tests in animals and mitotane-treated patients. Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences, 2006; 837(1-2): 69-75. https://doi.org/10.1016/j.jchromb.2006.04.005 ☒
Djerada Z, Feliu C, Tournois C, Vautier D, Binet L, Robinet A, Gozalo C, Lamiable D and
☒ Millart H. Validation of a fast method for quantitative analysis of elvitegravir, raltegravir, maraviroc, etravirine, tenofovir, boceprevir and 10 other antiretroviral agents in human plasma samples with a new UPLC-MS/MS technology. Journal of Pharmaceutical and Biomedical Analysis, 2013; 86, 100-11.
☒
https://doi.org/10.1016/j.jpba.2013.08.002
European Medicines Agency -. (s. d.).
☒ http://www.ema.europa.eu/ema/index.jsp?curl=pages/includes/document/document_d etail.jsp?webContentId=WC500109686%26mid=WC0b01ac058009a3dc European Medicines Agency - Find medicine - Lysodren.
http://www.ema.europa.eu/ema/index.jsp?curl=pages/medicines/human/medicines/00 0521/human_med_000895.jsp&mid=WC0b01ac058001d124
☒
Faggiano A, Leboulleux S, Young J, Schlumberger M and Baudin E. Rapidly progressing ☒ high o,p’DDD doses shorten the time required to reach the therapeutic threshold with an acceptable tolerance: preliminary results. Clinical Endocrinology 2006: 64(1), 110-3. https://doi.org/10.1111/j.1365-2265.2005.02403.x
Garg MB, Sakoff JA and Ackland SP. A simple HPLC method for plasma level monitoring
☒ of mitotane and its two main metabolites in adrenocortical cancer patients. Journal of
Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences
☒ 2011; 879(23), 2201-5. https://doi.org/10.1016/j.jchromb.2011.06.001
Hermsen IG, Fassnacht M, Terzolo M, Houterman S, den Hartigh J, Leboulleux S, Daffara F, ☒ Berruti A, Chadarevian R, Schlumberger M, Allolio B, Haak HR and Baudin E. Plasma concentrations of o,p’DDD, o,p’DDA, and o,p’DDE as predictors of tumor response to mitotane in adrenocortical carcinoma: results of a retrospective ENS@T ☒
multicenter study. The Journal of Clinical Endocrinology and Metabolism, 2011;
☒
96(6), 1844-51. https://doi.org/10.1210/jc.2010-2676
Hescot S, Paci A, Seck A, Slama A, Viengchareun S, Trabado S, Brailly-Tabard S, Al
☒
Ghuzlan A, Young, Baudin E and Lombès M. (2014). The lack of antitumor effects of o,p’DDA excludes its role as an active metabolite of mitotane for adrenocortical carcinoma treatment. Hormones & Cancer 2014; 5(5), 312- 23.
https://doi.org/10.1007/s12672-014-0189-7
Inouye M, Mio T and Sumino K. Use of GC/MS/SIM for rapid determination of plasma levels of o,p’-DDD, o,p’-DDE and o,p’-DDA. Clinica Chimica Acta; International Journal of Clinical Chemistry, 1987; 170(2-3), 305-14.
☒
Kasperlik-Zaluska AA and Cichocki A. Clinical role of determination of plasma mitotane and its metabolites levels in patients with adrenal cancer: results of a long-term follow-up. Journal of Experimental Therapeutics & Oncology 2005; 5(2), 125-32.
☒
Mauclère-Denost S, Leboulleux S, Borget I, Paci A, Young J, Al Ghuzlan A, Deandreis D, Drouard L, Tabarin A, Chanson P Martin Schlumberger M and Baudin E. High- dosemitotane strategy in adrenocortical carcinoma: prospective analysis of plasma mitotane measurement during the first 3 months of follow-up. European Journal of Endocrinology / European Federation of Endocrine Societies, 2012, 166(2), 261- 8. https://doi.org/10.1530/EJE-11-0557
Mornar A, Sertić M, Turk N, Nigović B and Koršić M. Simultaneous analysis of mitotane and its main metabolites in human blood and urine samples by SPE-HPLC technique. Biomedical Chromatography: BMC, 2012; 26(11), 1308-14. ☒
☒ https://doi.org/10.1002/bmc.2696
Paci A, Veal G, Bardin C, Levêque D, Widmer N, Beijnen J, Astier A and Chatelut E. Review of therapeutic drug monitoring of anticancer drugs part 1 — cytotoxics.
European Journal of Cancer (Oxford, England: 1990), 2014; 50(12), 2010-9.
☒ https://doi.org/10.1016/j.ejca.2014.04.014
PDQ Adult Treatment Editorial Board. Adrenocortical Carcinoma Treatment (PDQ®):
☒ Health Professional Version. In PDQ Cancer Information Summaries. Bethesda (MD): National Cancer Institute (US) 2002
http://www.ncbi.nlm.nih.gov/books/NBK65956/
Terzolo M, Pia A, Berruti A, Osella G, Alì A, Carbone V, … and Angeli A. Low-dose monitored mitotane treatment achieves the therapeutic range with manageable side effects in patients with adrenocortical cancer. The Journal of Clinical Endocrinology and Metabolism, 2000; 85(6), 2234-8. https://doi.org/10.1210/jcem.85.6.6619
☒ ☒
Van Slooten H, Moolenaar AJ, Van Seters AP and Smeenk D. The treatment of
adrenocortical carcinoma with o,p’-DDD: prognostic implications of serum level monitoring. European Journal of Cancer & Clinical Oncology, 1984; 20(1), 47-53.
☒ Viswanathan CT, Bansal S, Booth B, DeStefano AJ, Rose MJ, Sailstad J, … and Weiner R. Quantitative bioanalytical methods validation and implementation: best practices for chromatographic and ligand binding assays. Pharmaceutical Research, 2007; 24(10), 1962-73. https://doi.org/10.1007/s11095-007-9291-7
Accepted
0.020
0.018
0.016-
0.014
0.012
0.010-
₹
0.008
0.006
0.004-
Mitotane - 7.336
0.002
0.000
1
4
-0.002
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
4.50
5.00
5.50
6.00
6.50
7.00
7.50
8.00
Figure 1A
Minutes
0.020
0.018-
0.016-
0.014
0.012-
0.010-
2
0.008
0.006-
Mitotane - 7.073
0.004
0.002-
+
0.000
4
4
-0.002
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
4.50
5.00
5.50
6.00
6.50
7.00
7.50
8.00
Figure 1B
Minutes
U.UZU
0.018-
0.016-
Mitotane - 7.105
0.014-
0.012
0.010-
2
0.008-
0.006
0.004
0.002
0.000
4
A
-0.002
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
4.50
5.00
5.50
6.00
6.50
7.00
7.50
8.00
Minutes
Figure 1C
Figure 1: Examples of chromatograms.
☒ Figure 1 A: Chromatogram of a blank plasma extract.
Figure 1 B: Chromatogram of mitotane: sample spiked at the concentration of 0.78 mg.L-1 (LLOQ)
Figure 1 C: Chromatogram of mitotane: sample spiked at the concentration of 5 mg.L-1
Accepted Arti®
0.30
0.25
o,p’-DDA
0.20
₴
3.433
o,p’-DDD
0.15
o,p’-DDE
0.10
7.228
9.391
0.05
0.00
A
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
10.00
11.00
Minutes
The figure shows the chromatogram peaks for o,p’-DDA, mitotane (o,p’-DDD) and o,p’- DDE at the concentration of 10 mg.L- at retention times 3.4, 7.2 and 9.3 minutes respectively.
Accept
25
20-
20
Concentration (mg/L)
1
2
20
Concentration (mg/L)
Concentration (mg/L)
18
15-
15
16-
10-
10
5-
14-
5-
0-
12-
0-
0
2
4
6
8
0
5
10
15
0
5
10
15
20
25
Time (months)
Time (months)
Time (months)
Figure 3A
Figure 3B
Figure 3C
Accepted
| icle® | Intra-assay (n= 6) | Inter-assay (n=30) | ||
|---|---|---|---|---|
| Precision (RSD %) | Accuracy (%) | Precision (RSD %) | Accuracy (%) | |
| QCL (5 mg.L-1) | 1.08 | 107.43 | 3.78 | 100.83 |
| QCM (10 mg.L-1) | 0.48 | 103.18 | 3.12 | 102.02 |
| QCH (20 mg.L-1) | 0.83 | 113.69 | 6.32 | 105.59 |
| LLOQ (0.78 mg.L-1) | 6.00 | 112.61 | 4.62 | 114.74 |
| ULOQ (25 mg.L-1) | 4.02 | 95.65 | 4.79 | 93.75 |
RSD: relative standard deviation.
Accepted
| QC1 | QC2 | QC3 | QC4 | QC 5 | QC 6 | |
|---|---|---|---|---|---|---|
| Our values | 25.10 | 4.70 | 13.66 | 12.39 | 4.80 | 10.80 |
| Means values between laboratories | 26.18 | 5.54 | 15.11 | 12.74 | 4.90 | 8.80 |
| Theorical values | 25.00 | 5.00 | patient sample | 12.50 | patient sample | 10.00 |
| Accuracy (%) | 100.40 | 94.00 | 90.00 | 99.12 | 98.00 | 103.00 |
RSD: relative standard deviation.
Accepted
Table 3: Comparison of the different procedures for mitotane quantification
| References | Benecke et al, 1987 | Inouye et al , 1987 | Andersen et al, 1999 | De Francia et al, 2006 | Garg et al, 2011 | Mornar et al, 2012 | Our method |
| Technique | Gas chromatography coupled with electron capture detection | Gas chromatography coupled mass spectrometry | Liquid chromatography coupled with ultra violet detection | Liquid chromatography coupled with ultra violet detection | Liquid chromatography coupled with ultra violet detection | Liquid chromatography coupled with ultra violet detection | Liquid chromatography coupled with ultra violet detection |
| Sample volume, | 50 µL | 100 µL | 500 µL | 200 ML | 500 µL | 100 µL | |
| Sample preparation | - extraction with heptane | - absorption on a filter paper - extraction with benzene - derivation | - precipitation of plasma proteins with acetonez | - extraction with acetone - SPE for o,p'-DDA - dilution in mobile phase 50/50 | - precipitation with cold ethanol - addition of KH2PO4 50 mM buffer pH2.5 to supernatant | - liquid extraction with acetonitrile - reversed-phase solid- phase extraction cartridges (Discovery DSC-18, 500mg, 3mL, Supelco) | - precipitation of plasma proteins with methanol |
| Chromatographic conditions | - 30-m (film thickness of 1.5 pm) fused silica mega bore column DB-1 - helium, - flow rate : 1.5 ml/mm. - column temperature 220 °℃ | - isocratic conditions on a silica-based diphenyl column | -C18 reversed-phase column (Lichrocart250-4 Lichrospher 100 RP-18, 5um, VWR, Germany) preceded by a guard column (VWR) -Mobile phase : gradient of water, methanol, acetonitrile (0-6.5 min: 10:10:80, v/v/v; 6.6-9.7 min:5:5:90, v/v/v; 9.7-15 min: 10:10:80, v/v/v) at the - flow rate of 1 ml/min at 35℃ (different conditions for o,p'-DDA) | -Isocratic elution of the mobile phase, methanol: 50 mM KH2PO4 buffer pH2.5 (71:29,v/v) - flow rate of 0.6 ml/min - Waters Nova-Pak Phenyl column (150mm×3.9mm I.D.,4 um particle size) kept at 57℃ preceded by Waters Nova-Pak Phenyl (3.9mm×20mm,4m) guard column. | - column Symmetry C18,150 4.6mm, particle size 3.5um (Water) kept at 25℃ - mobile phase : acetonitrile, ultrapure water and formic acid at the ratio of 90:10:0.1 (v/v/v). - flow rate 1mL/min | - Uptisphere 5 µM ODB pre-column from Interchim, LICHROSPHER 100 RP-8 (5 um, 250 x 4 mm) column maintained at 25 °C. - mobile phase : phosphate buffer pH 3 and acetonitrile, ratio of 25:75 (v/v). - Isocratic flow rate 1.2 mL.min-1 | |
| Internal standard | Yes | Yes | No | Yes | Yes | Yes | No |
| Analysis time | 14 minutes | 15 minutes | 21 minutes | 10 minutes | 8 minutes | ||
| Detector | - source temperature 250"C, - ionization voltage 70 eV, - ionization current 30 PA - acceleration energy 3.0 KV. - m/z : 199, 210, 235 and 246 | 218 nm | 218 nm | 226 nm | 230 nm | 230 nm | |
| Compounds | o,p'-DDD and o,p'-DDE | o,p'-DDD, o,p'-DDE and o,p'-DDA | o,p'-DDD, o,p'-DDE and o,p'-DDA | o,p'-DDT, o,p'-DDD,, o,p'- DDE and o,p'-DDA | o,p'-DDD, o,p'-DDE and o,p'- DDA | o,p'-DDD, o,p'-DDE and o,p'-DDA | o,p'-DDD |
| Method validation | - recovery 87% - RSD < 5% | - recovery - precision - limit of quantification - stability | - recovery - precision - limit of quantification | - linearity, - precision - recovery | - precision - accuracy - specificity - selectivity | - selectivity - linearity - extraction efficiency, - precision - accuracy | - linearity - limit of quantification - precision - accuracy - specificity |
| - limits of detection and quantification - stability | - selectivity - stability - carry over | ||||||
|---|---|---|---|---|---|---|---|
| Accepted Article Calibration range for o,p'-DDD | 1-100 mg/L | 0,5-800 μΜ | 0,5-40 mg/L | 1-50 mg/L | 0.5 -50 mg/L | 0.78 to 25 mg/L |