Journal of Pharmaceutical and Biomedical Analysis 95 (2014) 107–112
Contents lists available at ScienceDirect
Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba
Short communication
Quantification of trabectedin in human plasma: Validation of a high-performance liquid chromatography–mass spectrometry method and its application in a clinical pharmacokinetic study Monique Zangarini a , Laura Ceriani a , Federica Sala a , Elena Marangon c , Renzo Bagnati b , Maurizio D’Incalci a , Federica Grosso d , Massimo Zucchetti a,∗ a IRCCS-Istituto di Ricerche Farmacologiche “Mario Negri”, Laboratory of Cancer Pharmacology, Department of Oncology, Via G. La Masa 19, 20156 Milan, Italy b IRCCS-Istituto di Ricerche Farmacologiche “Mario Negri”, Department of Environmental Health, Via G. La Masa 19, 20156 Milan, Italy c Centro di Riferimento Oncologico (CRO), National Cancer Institute, Aviano (PN), Italy d Oncology, SS Antonio e Biagio General Hospital, Via Venezia 16, Alessandria, Italy
a r t i c l e
i n f o
Article history: Received 20 January 2014 Received in revised form 21 February 2014 Accepted 22 February 2014 Available online 2 March 2014 Keywords: Trabectedin HPLC–MS/MS Human plasma Pharmacokinetics Liposarcoma
a b s t r a c t A rapid, sensitive and specific HPLC–MS/MS method was developed and validated for the quantification of trabectedin in human plasma after using deuterated trabectedin as Internal Standard (IS). After the addition of ammonium sulphate, the analyte was extracted from human plasma with acidified methanol (0.1 M HCl). Chromatographic separation was done on an Accucore XL C18 column (4 m; 50 mm × 2.1 mm) using a Mobile Phase (MP) consisting of CH3 COONH4 10 mM, pH 6.8 (MP A) and CH3 OH (MP B). The mass spectrometer worked with electrospray ionization in positive ion mode and Selected Reaction Monitoring (SRM), using target ions at [M−H2 O+H]+ m/z 744.4 for trabectedin and [M−H2 O+H]+ m/z 747.5 for the IS. The standard curve was linear (R2 ≥ 0.9955) over the concentration range 0.025–1.0 ng/ml and had good back-calculated accuracy and precision. The intra- and inter-day precision and accuracy determined on three quality control samples (0.04, 0.08 and 0.80 ng/ml) were 81% and the lower limit of quantification 0.025 ng/ml. The method was successfully applied to study trabectedin pharmacokinetics in a patient with a liposarcoma who received 1.3 mg/m2 as a 24 h continuous i.v. infusion. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Trabectedin (Fig. 1) is an anticancer agent whose chemical structure is characterized by three fused tetrahydroisoquinoline rings; it is registered in Europe and in several other countries for the second-line treatment of soft tissue sarcoma and in combination with liposomal doxorubicin for ovarian cancer [1,2]. Trabectedin was originally extracted from a marine organism, the tunicate Ecteinascidia turbinata, and is now produced synthetically. It binds in the DNA minor groove causing structural and functional changes in DNA, leading to inhibition of transcription that is gene- and promoter-dependent. Recently it has been reported that part of its antitumor activity is related to its ability to affect tumor associated macrophage, thus modifying tumor microenvironment [1].
∗ Corresponding author. Tel.: +39 02 39014549; fax: +39 02 39014734. E-mail address: [email protected] (M. Zucchetti). http://dx.doi.org/10.1016/j.jpba.2014.02.018 0731-7085/© 2014 Elsevier B.V. All rights reserved.
To study the clinical pharmacokinetics of trabectedin we developed a highly sensitive, accurate and reproducible method for quantification of the drug in human plasma. To date there are some methods for the analysis of trabectedin in human plasma, based on liquid chromatography (LC) [3–5]. The first method [3] employed conventional LC-UV, reaching a Lower Limit of Quantification (LLOQ) of 1 ng/ml in plasma using a 500 l sample and solid phase extraction (SPE). This method was later significantly improved by using LC coupled with electrospray ionization tandem mass spectrometry, achieving a LLOQ of 0.01 ng/ml [4]. The third method [5] uses 100 l of plasma and is based on column switching extraction and tandem mass spectrometry detection that give a LLOQ of 0.05 ng/ml. For further bioanalytical studies see [6–8]. We describe a new method validated according to FDA–EMA guidance [9,10]. It is currently being used in a pharmacokinetic study on cancer patients in a clinical trial. The assay requires only 100 l of plasma, very simple extraction with acidified methanol and is faster and less expensive than SPE. It is based on liquid
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M. Zangarini et al. / Journal of Pharmaceutical and Biomedical Analysis 95 (2014) 107–112
Fig. 1. Chemical structures of trabectedin (A) and trabectedin d3 (B).
2. Experimental
plasma (90 L) to obtain the following plasma concentrations: 0.04, 0.08 and 0.8 ng/ml. The calibration curve samples and QCs were processed as described below.
2.1. Standards and chemicals
2.4. Processing samples
Analytical reference standards of trabectedin (MW 761.84 g/mol) and deuterated trabectedin ([2 H3 ] trabectedin; MW 764.84 g/mol), used as Internal Standard (IS) were kindly provided by Pharmamar (Colmenar Viejo, Madrid, Spain). HPLC grade methanol, analytical grade ammonium acetate (CH3 COONH4 ) and ammonium sulphate were purchased from Sigma–Aldrich Co. (Milan, Italy). Control human plasma with lithium heparin used to prepare daily standard calibration curves and quality control samples (QCs) was obtained from volunteers.
After thawing plasma samples at room temperature, 100 l of the actual sample, standard or QC sample were transferred to a 1.5 ml Eppendorf polypropylene tube and 10 l of the IS working solution was added; the mixture was vortexed, 50 l of ammonium sulphate (1.4 M) was added, vortexed again and then finally mixed with 400 l of 0.1 M HCl in methanol. Each tube was thoroughly vortexed for 30 s and centrifuged at 4 ◦ C for 10 min at 4000 × g; the obtained supernatant was then transferred to an Eppendorf polypropylene tube, dried under nitrogen at 35 ◦ C, and then reconstituted with 150 l of Mobile Phase A (MP A) and B (MP B) in the proportion 1:1, v/v. Then the tube was vortexed for 10 s and centrifuged at 4 ◦ C for 10 min at 4000 × g; finally the supernatant was transferred to an autosampler glass vial and 20 l were injected into the HPLC system.
chromatography–tandem mass spectrometry (LC–MS/MS) and is very sensitive, with a LLOQ of 0.025 ng/ml.
2.2. Standard and quality control solutions From a stock solution of 50.8 g/ml were prepared two trabectedin working solutions in methanol at the concentration of 100 ng/ml for both standards and quality controls. The stock solution for the IS was prepared at 10 g/ml in methanol. These solutions were stored at −20 ◦ C. A series of working solutions to prepare the plasma standard points of the calibration curve and the plasma quality control (Qc) samples were obtained by diluting the working solutions at 100 ng/ml with methanol in order to have the final trabectedin concentrations of: 0.25 ng/ml (E), 0.5 ng/ml (D), 1.0 ng/ml (C), 5.0 ng/ml (B), 10.0 ng/ml (A) for standards and 0.4 ng/ml (QcL), 0.8 ng/ml (QcM), 8.0 ng/ml (QcH) for quality controls. The IS working solution was prepared at 100 ng/ml by diluting the stock solution with methanol. 2.3. Standards and quality control samples A five-point plasma calibration curve was prepared freshly every day during the validation study. Each calibration sample was prepared by adding 10 l of each standard solution from E to A to 90 l of blank human plasma to obtain the following concentrations: 0.025, 0.05, 0.1, 0.5 and 1.0 ng/ml, the last being the Upper Limit of Quantification (ULOQ). Each calibration curve included a blank sample (plasma processed without IS) and a zero blank sample (plasma processed with the IS). Three QC samples were used for each concentration level. For each QC sample 10 l of the working quality control solutions (L, M, H) were added to blank human
2.5. Chromatographic conditions The HPLC system consisted of a Series 200 auto-sampler and micro-pump (Perkin Elmer, Waltham, MA, USA). Samples were separated on an Accucore XL C18 chromatographic column (4 m; 50 mm × 2.1 mm), coupled with a pre-column with the same material, both thermostatically controlled at 35 ◦ C and provided by Thermo Scientific (Milan, Italy). The Mobile Phases (MP) for chromatographic separation were CH3 COONH4 10 mM, pH 6.8 in bidistilled water (MP A) and CH3 OH (MP B). The HPLC system was set up with a flow rate of 0.2 ml/min and the following linear gradient: step 1: initial condition from 90% MP A to 5% in 6 min; step 2: constant for 1 min; step 3: from 5% MP A to the initial condition over 1 min; step 4: reconditioning 6 min. The total run time was 14 min. 2.6. Mass spectrometry The HPLC system was coupled with an API 4000 triple quadrupole mass spectrometer AB SCIEX (Toronto, Canada). Standard solutions (50 ng/ml) of trabectedin and IS were infused at a flow rate of 10 L/min in order to optimize all the MS parameters. Positive ion mode was used to obtain the mass spectra (MS1 ) and the product ion spectra (MS2 ). The instrument was equipped
M. Zangarini et al. / Journal of Pharmaceutical and Biomedical Analysis 95 (2014) 107–112
with a TurboIonSpray source operated at 500 ◦ C and with ion spray voltage of 5000 V. The biological samples were analyzed with electrospray ionization (ESI), using zero air as nebulizer gas (35 psi) and as heater gas (50 psi). Nitrogen was employed as curtain gas (20 psi) and as collision gas at 5 psi (CAD). Quantification was in Selected Reaction Monitoring (SRM) mode with the following transitions: m/z 744.4 → 495.4 for trabectedin and m/z 747.5 → 253.3 for the IS. Data was processed with Analyst 1.5.1 software package (AB SCIEX). 2.7. Validation This study was conducted in accordance with the European Medicines Agency and the Food Drug and Administration guidance on bio-analytical method validation [9,10]. The method was validated by examining the following parameters: recovery (in agreement with FDA), linearity, intra- and inter-day precision and accuracy, and Lower Limit of Quantification (LLOQ), selectivity and matrix effect (in agreement with EMA). 2.7.1. Recovery The percentage recovery was determined by comparing the peak area of the analyte extracted from plasma QC samples, prepared at three different concentrations of trabectedin (0.04, 0.08 and 0.8 ng/ml), with the peak area of the extracted matrix prepared in five replicates and added with the same amount of
109
the analyte. The recovery of IS was calculated the same way at a concentration of 10.0 ng/ml [9]. 2.7.2. Linearity The linearity of calibration curves was validated over five working days with calibration curves prepared as described in Section 2.3. For each standard point, the ratio of the HPLC–MS/MS peak area for trabectedin to the IS was calculated and plotted against the nominal concentration of trabectedin in the sample. The linearity of the standard curves was checked by regression analysis and the goodness of the regression by calculating Pearson’s determination coefficient R2 and by comparison of the true and back-calculated concentrations of the calibration standards. The accuracy of backcalculated values of an individual point had to be within 85–115% of the theoretical concentration (80–120% at the LLOQ), and all the standards had to meet these criteria, including the LLOQ and highest calibrator, ULOQ [9,10]. 2.7.3. Intra-day and inter-day precision and accuracy Precision and accuracy were checked on five days by measuring the analyte in three replicates at three QC levels at the nominal concentrations of 0.04, 0.08 and 0.8 ng/ml. To analyze the QCs, different standard calibration curves were plotted and processed on each of the five days of the validation study. The precision of the method at each concentration was reported as the coefficient of variation (CV%), expressing the standard deviation as a percentage
Fig. 2. MS and MS/MS mass spectra of trabectedin and IS (trabectedin d3 ).
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M. Zangarini et al. / Journal of Pharmaceutical and Biomedical Analysis 95 (2014) 107–112
of the mean calculated concentration; the accuracy was determined by expressing the mean calculated concentration as a percentage of the nominal concentration. In each run, the concentration measured for at least six of the nine QC samples had to be within 15% of the nominal value: at each concentration level, only one QC sample could be excluded [9,10]. 2.7.4. Limit of quantification, selectivity and matrix effect The LLOQ of the bioanalytical method was the concentration of the lowest standard and was assessed by adding trabectedin to an aliquot of a pool of blank human plasma to obtain a final concentration of 0.025 ng/ml. Selectivity was proved using six independent sources of blank human plasma, which are individually analyzed and evaluated for interference; a single 100 l aliquot from each of the six matrices was spiked with the drug at the LLOQ [9,10]. Both limit of quantification and selectivity had to have acceptable accuracy (
Contents lists available at ScienceDirect
Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba
Short communication
Quantification of trabectedin in human plasma: Validation of a high-performance liquid chromatography–mass spectrometry method and its application in a clinical pharmacokinetic study Monique Zangarini a , Laura Ceriani a , Federica Sala a , Elena Marangon c , Renzo Bagnati b , Maurizio D’Incalci a , Federica Grosso d , Massimo Zucchetti a,∗ a IRCCS-Istituto di Ricerche Farmacologiche “Mario Negri”, Laboratory of Cancer Pharmacology, Department of Oncology, Via G. La Masa 19, 20156 Milan, Italy b IRCCS-Istituto di Ricerche Farmacologiche “Mario Negri”, Department of Environmental Health, Via G. La Masa 19, 20156 Milan, Italy c Centro di Riferimento Oncologico (CRO), National Cancer Institute, Aviano (PN), Italy d Oncology, SS Antonio e Biagio General Hospital, Via Venezia 16, Alessandria, Italy
a r t i c l e
i n f o
Article history: Received 20 January 2014 Received in revised form 21 February 2014 Accepted 22 February 2014 Available online 2 March 2014 Keywords: Trabectedin HPLC–MS/MS Human plasma Pharmacokinetics Liposarcoma
a b s t r a c t A rapid, sensitive and specific HPLC–MS/MS method was developed and validated for the quantification of trabectedin in human plasma after using deuterated trabectedin as Internal Standard (IS). After the addition of ammonium sulphate, the analyte was extracted from human plasma with acidified methanol (0.1 M HCl). Chromatographic separation was done on an Accucore XL C18 column (4 m; 50 mm × 2.1 mm) using a Mobile Phase (MP) consisting of CH3 COONH4 10 mM, pH 6.8 (MP A) and CH3 OH (MP B). The mass spectrometer worked with electrospray ionization in positive ion mode and Selected Reaction Monitoring (SRM), using target ions at [M−H2 O+H]+ m/z 744.4 for trabectedin and [M−H2 O+H]+ m/z 747.5 for the IS. The standard curve was linear (R2 ≥ 0.9955) over the concentration range 0.025–1.0 ng/ml and had good back-calculated accuracy and precision. The intra- and inter-day precision and accuracy determined on three quality control samples (0.04, 0.08 and 0.80 ng/ml) were 81% and the lower limit of quantification 0.025 ng/ml. The method was successfully applied to study trabectedin pharmacokinetics in a patient with a liposarcoma who received 1.3 mg/m2 as a 24 h continuous i.v. infusion. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Trabectedin (Fig. 1) is an anticancer agent whose chemical structure is characterized by three fused tetrahydroisoquinoline rings; it is registered in Europe and in several other countries for the second-line treatment of soft tissue sarcoma and in combination with liposomal doxorubicin for ovarian cancer [1,2]. Trabectedin was originally extracted from a marine organism, the tunicate Ecteinascidia turbinata, and is now produced synthetically. It binds in the DNA minor groove causing structural and functional changes in DNA, leading to inhibition of transcription that is gene- and promoter-dependent. Recently it has been reported that part of its antitumor activity is related to its ability to affect tumor associated macrophage, thus modifying tumor microenvironment [1].
∗ Corresponding author. Tel.: +39 02 39014549; fax: +39 02 39014734. E-mail address: [email protected] (M. Zucchetti). http://dx.doi.org/10.1016/j.jpba.2014.02.018 0731-7085/© 2014 Elsevier B.V. All rights reserved.
To study the clinical pharmacokinetics of trabectedin we developed a highly sensitive, accurate and reproducible method for quantification of the drug in human plasma. To date there are some methods for the analysis of trabectedin in human plasma, based on liquid chromatography (LC) [3–5]. The first method [3] employed conventional LC-UV, reaching a Lower Limit of Quantification (LLOQ) of 1 ng/ml in plasma using a 500 l sample and solid phase extraction (SPE). This method was later significantly improved by using LC coupled with electrospray ionization tandem mass spectrometry, achieving a LLOQ of 0.01 ng/ml [4]. The third method [5] uses 100 l of plasma and is based on column switching extraction and tandem mass spectrometry detection that give a LLOQ of 0.05 ng/ml. For further bioanalytical studies see [6–8]. We describe a new method validated according to FDA–EMA guidance [9,10]. It is currently being used in a pharmacokinetic study on cancer patients in a clinical trial. The assay requires only 100 l of plasma, very simple extraction with acidified methanol and is faster and less expensive than SPE. It is based on liquid
108
M. Zangarini et al. / Journal of Pharmaceutical and Biomedical Analysis 95 (2014) 107–112
Fig. 1. Chemical structures of trabectedin (A) and trabectedin d3 (B).
2. Experimental
plasma (90 L) to obtain the following plasma concentrations: 0.04, 0.08 and 0.8 ng/ml. The calibration curve samples and QCs were processed as described below.
2.1. Standards and chemicals
2.4. Processing samples
Analytical reference standards of trabectedin (MW 761.84 g/mol) and deuterated trabectedin ([2 H3 ] trabectedin; MW 764.84 g/mol), used as Internal Standard (IS) were kindly provided by Pharmamar (Colmenar Viejo, Madrid, Spain). HPLC grade methanol, analytical grade ammonium acetate (CH3 COONH4 ) and ammonium sulphate were purchased from Sigma–Aldrich Co. (Milan, Italy). Control human plasma with lithium heparin used to prepare daily standard calibration curves and quality control samples (QCs) was obtained from volunteers.
After thawing plasma samples at room temperature, 100 l of the actual sample, standard or QC sample were transferred to a 1.5 ml Eppendorf polypropylene tube and 10 l of the IS working solution was added; the mixture was vortexed, 50 l of ammonium sulphate (1.4 M) was added, vortexed again and then finally mixed with 400 l of 0.1 M HCl in methanol. Each tube was thoroughly vortexed for 30 s and centrifuged at 4 ◦ C for 10 min at 4000 × g; the obtained supernatant was then transferred to an Eppendorf polypropylene tube, dried under nitrogen at 35 ◦ C, and then reconstituted with 150 l of Mobile Phase A (MP A) and B (MP B) in the proportion 1:1, v/v. Then the tube was vortexed for 10 s and centrifuged at 4 ◦ C for 10 min at 4000 × g; finally the supernatant was transferred to an autosampler glass vial and 20 l were injected into the HPLC system.
chromatography–tandem mass spectrometry (LC–MS/MS) and is very sensitive, with a LLOQ of 0.025 ng/ml.
2.2. Standard and quality control solutions From a stock solution of 50.8 g/ml were prepared two trabectedin working solutions in methanol at the concentration of 100 ng/ml for both standards and quality controls. The stock solution for the IS was prepared at 10 g/ml in methanol. These solutions were stored at −20 ◦ C. A series of working solutions to prepare the plasma standard points of the calibration curve and the plasma quality control (Qc) samples were obtained by diluting the working solutions at 100 ng/ml with methanol in order to have the final trabectedin concentrations of: 0.25 ng/ml (E), 0.5 ng/ml (D), 1.0 ng/ml (C), 5.0 ng/ml (B), 10.0 ng/ml (A) for standards and 0.4 ng/ml (QcL), 0.8 ng/ml (QcM), 8.0 ng/ml (QcH) for quality controls. The IS working solution was prepared at 100 ng/ml by diluting the stock solution with methanol. 2.3. Standards and quality control samples A five-point plasma calibration curve was prepared freshly every day during the validation study. Each calibration sample was prepared by adding 10 l of each standard solution from E to A to 90 l of blank human plasma to obtain the following concentrations: 0.025, 0.05, 0.1, 0.5 and 1.0 ng/ml, the last being the Upper Limit of Quantification (ULOQ). Each calibration curve included a blank sample (plasma processed without IS) and a zero blank sample (plasma processed with the IS). Three QC samples were used for each concentration level. For each QC sample 10 l of the working quality control solutions (L, M, H) were added to blank human
2.5. Chromatographic conditions The HPLC system consisted of a Series 200 auto-sampler and micro-pump (Perkin Elmer, Waltham, MA, USA). Samples were separated on an Accucore XL C18 chromatographic column (4 m; 50 mm × 2.1 mm), coupled with a pre-column with the same material, both thermostatically controlled at 35 ◦ C and provided by Thermo Scientific (Milan, Italy). The Mobile Phases (MP) for chromatographic separation were CH3 COONH4 10 mM, pH 6.8 in bidistilled water (MP A) and CH3 OH (MP B). The HPLC system was set up with a flow rate of 0.2 ml/min and the following linear gradient: step 1: initial condition from 90% MP A to 5% in 6 min; step 2: constant for 1 min; step 3: from 5% MP A to the initial condition over 1 min; step 4: reconditioning 6 min. The total run time was 14 min. 2.6. Mass spectrometry The HPLC system was coupled with an API 4000 triple quadrupole mass spectrometer AB SCIEX (Toronto, Canada). Standard solutions (50 ng/ml) of trabectedin and IS were infused at a flow rate of 10 L/min in order to optimize all the MS parameters. Positive ion mode was used to obtain the mass spectra (MS1 ) and the product ion spectra (MS2 ). The instrument was equipped
M. Zangarini et al. / Journal of Pharmaceutical and Biomedical Analysis 95 (2014) 107–112
with a TurboIonSpray source operated at 500 ◦ C and with ion spray voltage of 5000 V. The biological samples were analyzed with electrospray ionization (ESI), using zero air as nebulizer gas (35 psi) and as heater gas (50 psi). Nitrogen was employed as curtain gas (20 psi) and as collision gas at 5 psi (CAD). Quantification was in Selected Reaction Monitoring (SRM) mode with the following transitions: m/z 744.4 → 495.4 for trabectedin and m/z 747.5 → 253.3 for the IS. Data was processed with Analyst 1.5.1 software package (AB SCIEX). 2.7. Validation This study was conducted in accordance with the European Medicines Agency and the Food Drug and Administration guidance on bio-analytical method validation [9,10]. The method was validated by examining the following parameters: recovery (in agreement with FDA), linearity, intra- and inter-day precision and accuracy, and Lower Limit of Quantification (LLOQ), selectivity and matrix effect (in agreement with EMA). 2.7.1. Recovery The percentage recovery was determined by comparing the peak area of the analyte extracted from plasma QC samples, prepared at three different concentrations of trabectedin (0.04, 0.08 and 0.8 ng/ml), with the peak area of the extracted matrix prepared in five replicates and added with the same amount of
109
the analyte. The recovery of IS was calculated the same way at a concentration of 10.0 ng/ml [9]. 2.7.2. Linearity The linearity of calibration curves was validated over five working days with calibration curves prepared as described in Section 2.3. For each standard point, the ratio of the HPLC–MS/MS peak area for trabectedin to the IS was calculated and plotted against the nominal concentration of trabectedin in the sample. The linearity of the standard curves was checked by regression analysis and the goodness of the regression by calculating Pearson’s determination coefficient R2 and by comparison of the true and back-calculated concentrations of the calibration standards. The accuracy of backcalculated values of an individual point had to be within 85–115% of the theoretical concentration (80–120% at the LLOQ), and all the standards had to meet these criteria, including the LLOQ and highest calibrator, ULOQ [9,10]. 2.7.3. Intra-day and inter-day precision and accuracy Precision and accuracy were checked on five days by measuring the analyte in three replicates at three QC levels at the nominal concentrations of 0.04, 0.08 and 0.8 ng/ml. To analyze the QCs, different standard calibration curves were plotted and processed on each of the five days of the validation study. The precision of the method at each concentration was reported as the coefficient of variation (CV%), expressing the standard deviation as a percentage
Fig. 2. MS and MS/MS mass spectra of trabectedin and IS (trabectedin d3 ).
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M. Zangarini et al. / Journal of Pharmaceutical and Biomedical Analysis 95 (2014) 107–112
of the mean calculated concentration; the accuracy was determined by expressing the mean calculated concentration as a percentage of the nominal concentration. In each run, the concentration measured for at least six of the nine QC samples had to be within 15% of the nominal value: at each concentration level, only one QC sample could be excluded [9,10]. 2.7.4. Limit of quantification, selectivity and matrix effect The LLOQ of the bioanalytical method was the concentration of the lowest standard and was assessed by adding trabectedin to an aliquot of a pool of blank human plasma to obtain a final concentration of 0.025 ng/ml. Selectivity was proved using six independent sources of blank human plasma, which are individually analyzed and evaluated for interference; a single 100 l aliquot from each of the six matrices was spiked with the drug at the LLOQ [9,10]. Both limit of quantification and selectivity had to have acceptable accuracy (
