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A rapid and robust UHPLC-DAD method for the quantification of amphotericin B in human plasma
Journal of Pharmaceutical and Biomedical Analysis 138 (2017) 142–145
Contents lists available at ScienceDirect
Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba
A rapid and robust UHPLC-DAD method for the quantification of amphotericin B in human plasma Sebastiano Barco a , Alessia Zunino a , Antonio D’Avolio b , Laura Barbagallo a , Angelo Maffia a , Gino Tripodi a , Elio Castagnola c , Giuliana Cangemi a,∗ a
Clinical Pathology Laboratory Unit, Istituto Giannina Gaslini, Genoa, Italy Department of Medical Sciences, University of Turin, Laboratory of Clinical Pharmacology and Pharmacogenetics, Amedeo di Savoia Hospital, Turin, Italy c Infectious Disease Unit, Istituto Giannina Gaslini, Genoa, Italy b
a r t i c l e
i n f o
Article history: Received 7 December 2016 Received in revised form 23 January 2017 Accepted 24 January 2017 Available online 8 February 2017 Keywords: UHPLC Amphotericin B
a b s t r a c t Amphotericin B is an antifungal drug widely used in Intensive Care Units. Therapeutic drug monitoring (TDM) of amphotericin B is recommended for the assessment of toxicity surveillance and treatment optimization. In this paper we described the development and validation of a new Ultra High Performance Liquid Chromatography coupled to Diode Array Detection (UHPLC-DAD) method for the quantification of Amphotericin B in 200 L human plasma over a wide range of concentrations (0.125–10 mg/L). The new method has been validated following international guidelines on bioanalytical method validation and showed high selectivity, high accuracy and precision and high process efficiency. The new UHPLC-DAD method that we describe is robust, rapid, cost effective and suitable for application to the routine TDM analyses. © 2017 Published by Elsevier B.V.
1. Introduction Amphotericin B is a polyene antifungal agent with activity against molds and yeasts. The liposomal formulation of amphotericin B (LAmB) is widely used for the treatment or prophylaxis of invasive antifungal disease both in adults and in children, with a significant reduced toxicity respect the initial deoxycholate (DAmB) formulation, even if nephro and hepatotoxicity can still be observed [1,2]. Amphotericin B presents a concentration-dependent activity [3] and could be both fungistatic and fungicidal depending on the concentration of the drug in various body fluids and the susceptibility of specific fungus. Therapeutic drug monitoring (TDM) of LAmB could be recommended in special populations such as immunocompromised patients [4] and might be essential in patients in intensive care. Concentrations of LAmB can be measured using high performance liquid chromatography (HPLC), liquid chromatography–mass spectrometry (LC–MS/MS), or bioas-
Abbreviations: UHPLC, ultra high performance liquid chromatography; DAD, diode array detector. ∗ Corresponding author at: Clinical Pathology Laboratory Unit, Istituto Giannina Gaslini, Via Gerolamo Gaslini 5, 16147 Genova. E-mail address: [email protected] (G. Cangemi). http://dx.doi.org/10.1016/j.jpba.2017.01.048 0731-7085/© 2017 Published by Elsevier B.V.
says. The quantification method has a significant impact on what exactly is being measured (i.e. total amphotericin B, protein-bound drug, liposome-associated drug and freely circulating drug). Therefore, caution is required with the interpretation of drug concentrations. The critical step of each method is the extraction of amphotericin B from the liposome. A complete destruction of the liposome by using organic solvents and the release of the active drug needs to be obtained in order to correctly estimate the total concentration of amphotericin B within the matrix [1,5]. To date no commercially available kits for the measurement of amphotericin B by HPLC are currently available. Several HPLC methods have been described for the quantification of amphotericin B in human plasma [6–8], but they are scarcely applicable to routine LamB TDM because poorly validated and characterized by extensive sample preparation protocols [6] or long run times [7]. More recently LC–MS based methods have been published [9–11]. In this paper we describe a new Ultra High Performance Liquid Chromatography coupled to Diode Array Detection (UHPLC- DAD) method for the determination of total amphotericin B in plasma. The new method has been validated according to international guidelines on bioanalytical method validation [12,13] and has been successfully applied to pediatric patients
S. Barco et al. / Journal of Pharmaceutical and Biomedical Analysis 138 (2017) 142–145
2. Materials and methods
143
simultaneously measured at 384 and 407 nm (amphotericin B) and 304 nm (IS).
2.1. Reagents and chemicals 2.6. HPLC method validation Amphotericin B and natamycin (IS) were purchased from Sigma (Milan, Italy). Acetonitrile was purchased from Carlo Erba Reagenti (Milan, Italy). All solutions were prepared with HPLC grade water obtained from a Milli-Q Plus water purification system. HPLC mobile phases were filtered by using Millipore membrane filters [0.45 m] (Millipore, Vimodrone MI, Italy). 2.2. Human samples Plasma was obtained from peripheral blood collected in sodium heparin-containing tubes by centrifuging at 4000 g for 5 min. Plasma samples were stored at −20 ◦ C until analysed. For method validation purposes, blank samples were obtained from healthy adult volunteers who were not being treated with Amphotericin B. Leftover samples derived from seven pediatric patients (age 2 months-18 years) of Intensive Care Units of G. Gaslini Institute who were being treated with Amphotericin B (Ambisome, LamB Gilead Sciences, Paris, France) for the treatment of invasive micoses were obtained. In order to test selectivity, leftover samples derived from 50 pediatric patients (age 6 months-10 years) of Intensive Care Units of G. Gaslini Institute who were not being treated with LamB but who were treated with the same co-medications were obtained. The Internal Review Board was notified about TDM program, but no formal approval was required since no additional blood sampling was required in order to set up the new method. 2.3. Sample preparation A 200 L aliquot of plasma was protein precipitated with 50 L of ZnSO4 0.1 M and 200 L of acetonitrile after addition of 20 L IS (20 mg/L). After vortexing, samples were centrifuged for 10 min at 13.000 × g at 4 ◦ C and the supernatant was transferred to autosampler vials and 10 L were injected in the UHPLC system. 2.4. Preparation of calibrators and quality control samples Stock solutions of 1 g/L amphotericin B in DMSO and 0.53 g/L IS in methanol, were prepared and stored at −20 ◦ C. Calibrators and quality controls (QC) were obtained by serial dilution of stock solutions with plasma obtained from healthy adult volunteers. A 7-point calibration curve was assessed using amphotericin B concentrations of 0.125–0.25–0.5–1–2.5–5–10 mg/L. QC samples were prepared at the following four concentration levels: 0.125 mg/L (LLOQ), 0.375 mg/L (low QC), 1.5 mg/L (medium QC) and 7.5 mg/L (high QC). 2.5. Chromatographic conditions Chromatography was performed by using an Ultimate 3000 UHPLC system (ThermoScientific, Milan, Italy) equipped with a diode array detector (DAD, Ultimate 300RS, Thermoscientific, Milan, Italy). Analysis was performed on a Hypersil Gold column (50 mm × 2.1 mm; particle size, 1.9 m; Thermoscientific, Milan, Italy). Analytes were separated by gradient elution at 50 ◦ C with mobile phase A consisting in 0.1 M KH2 PO4 in H2 O (adjusted at pH = 3 with H3 PO4 ) and mobile phase B of acetonitrile at a flow-rate of 0.8 mL/min. The percentage of mobile phase B was maintained at 10% for 0.2 min and then programmed to reach 100% in 5 min and then maintained 1.5 min. The column was finally reconditioned at 10% B in 1.5 min for a total run time of 8 min. The absorbances were
A laboratory scheme based on EMA and FDA guidelines on bioanalytical method validation [10,11] was used for assay validation. 2.6.1. Selectivity Selectivity was evaluated as a lack of interference of matrix or other concomitant medications by analyzing six different batches of pooled plasma from healthy volunteers. Moreover 50 samples derived from leftover plasma of patients of Institute G. Gaslini in Intensive Cure Units who were not being treated with amphotericin B but who were treated with several co-medications. Interfering components were considered as absent when the signal was less than 20% of the LLOQ for the analytes and less than 5% for the IS. 2.6.2. Carry- over Carry-over was assessed by injecting blank samples in triplicate after the highest calibration standard. The signal in the blank sample following the high standard should not be greater than 20% of the LLOQ and 5% for the IS. 2.6.3. Accuracy and precision Within-day and between-day precision and accuracy were determined at four concentration levels, using the four level QC samples (LLOQ, low, medium and high QC) and by analyzing each sample six separate times. Inter-day precision was estimated by repeating the procedure three times on three separate days. Accuracy was expressed as the mean relative error (percentage of agreement between calculated and nominal concentration of QCs). Precision was expressed as the coefficient of variation (CV%). The acceptance criteria for within- and between-day precision were ≤15% and for accuracy was 85–115% of the nominal concentrations. 2.6.4. Lower limit of quantification (LLOQ) The LLOQ was determined as the lowest sample concentration that provided measurements with an precision ≤20% and accuracy within 80–120% of the nominal concentration. 2.6.5. Linearity Linearity was evaluated by analyzing calibration standards three times on three separate days in the test range 0.125–10 mg/L. The slope, intercept, and correlation coefficient for each calibration curve were obtained by plotting the peak area ratio of analyte IS vs. the nominal concentration of each calibrator. The acceptance criteria for the amounts of back-calculated standard was ± 15% of the theoretical value (except ± 20% for the lowest standard). 2.6.6. Dilution integrity Dilution integrity was investigated by spiking a pool of plasma with amphotericin B at 18 mg/L. After serial dilution 1:2 and 1:4 with blank matrix, each sample was analysed five times. The acceptance criteria were ± 15% of the theoretical value. 2.6.7. Matrix effects and extraction recoveries Matrix effects and extraction recoveries were assessed at two different levels (corresponding to the QC low and QC high ) analysed in triplicate using 6 different batches of pooled plasma. Matrix effects were assessed by comparing peak area of amphotericin B spiked after extraction from plasma to peak area of pure solutions at the same concentration. Extraction recoveries were determined by comparing peak area of amphotericin B spiked before the extraction procedure to peak area of amphotericin B spiked after protein precipitation. Process efficiency was determined by comparing peak
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S. Barco et al. / Journal of Pharmaceutical and Biomedical Analysis 138 (2017) 142–145
Fig. 1. Chromatograms for amphotericin B. Panel A: IS (304 nm); Panel B: amphotericin B (384 nm); Panel C: amphotericin B (407 nm).
Table 1 Precision and accuracy results. Mean±SD (mg/L)
Precision (CV%)
Accuracy (R.E.%)
Mean±SD (mg/L)
Intra-day LLOQ QC low QC medium QC high
0.125 ± 0.02 0.375 ± 0.06 1.5 ± 0.25 7.5 ± 0.75
Precision (CV%)
Accuracy (R.E.%)
5.3 5.6 6.5 4.8
97.6 94.9 94.6 97.3
Inter-day 2.1 2.1 0.9 1.2
0.44 ± 0.02 1.8 ± 0.10 6.43 ± 0.59 26.28 ± 1.55
96.5 98.5 99.2 99.6
Table 2 Matrix effect and extraction recoveries.
Table 3 Stability results.
QC level
Extraction recoveries
Matrix effect
Processefficiency
QC low QC high
100 102.4
103.2 92.8
129.6 119.2
area of amphotericin B spiked before the extraction procedure to peak area of pure solution. Results were considered acceptable with CV < 15%.
Storage Condition
3. Results 3.1. Method development The extraction protocol based on protein precipitation with ZnSO4 and acetonitrile from 200 L plasma allowed a fast and efficient extraction of LamB. The use of Hypersil Gold 1.9 m column allowed us to obtain an excellent peak resolution with a short chromatographic separa-
QC high a
◦
24 h at 25 C 4 h at 25 ◦ C freeze/thaw 3 times 15 days at −20 ◦ C 15 days at +4 ◦ C extracted 3days at 25 ◦ C a
2.6.8. Stability Stability of extracted or unextracted samples was determined on QC low and QC high analysed in triplicate. Short-term temperature stability was determined by maintaining the samples at room temperature for 6 and 24 h. Long term temperature stability was determined by maintaining the samples at −20 ◦ C for 15 days or at +4 ◦ C for 7 or 15 days respectively. Freeze and thaw stability was determined after three freeze and thaw cycles. Stability of extracted samples was determined by maintaining extracts at room temperature for 72 h. Amphotericin B and IS stock solutions stability was determined by maintaining them at −20 ◦ C for 15 or 30 days.
QC low CV (%)
Recovery
CV (%)a
Recovery
4.4 1.1 2.0 1.0 0.5 3.0
93 99 98 99 97 96
9.0 8.0 3.0 0.5 1.0 2.0
98 98 99 99 98 91
CV calculated from three replicates.
tion (with a total run time of 8 min) thus greatly improving the productivity of the method and decreasing mobile phase usage. Chromatograms obtained for both amphotericin B and its IS are shown in Fig. 1. Retention times are 3.57 and 3.12 min for amphotericin B and its IS respectively. 3.2. Method validation The method was validated following international guidelines and showed excellent selectivity with no interfering peaks at the specified chromatographic conditions. Carry-over was absent. The LLOQ was 0.125 mg/L. The linear regression fit for the calibration curves was achieved. All back-calculated values did not differ from ±15% of the theoretical value. The results of the intra-inter assay precision and accuracy and recoveries were all inside the acceptable ranges (Table 1). The dilution integrity assay gave results always under ±15% of the theoretical value at the two dilution factors tested. Matrix effect, extraction recovery and process efficiency tests gave results inside the acceptable ranges (Table 2). Stability tests performed on QCs
S. Barco et al. / Journal of Pharmaceutical and Biomedical Analysis 138 (2017) 142–145
(Table 3) demonstrated that unprocessed samples were stable for at least 24 h at room temperature and for 15 days at 4 ◦ C and at −20 ◦ C and after three freeze/thaw cycles. Processed samples can be left at room temperature for three days without significant losses. The new UHPLC method has been tested on 9 samples derived from 7 pediatric patients treated with amphotericin B. The results were in the range 0.2-5.9 mg/L. 4. Discussion Several HPLC methods [6–8] have been previously described for the quantification of LAmB in human plasma. HPLC methods generally required time-consuming sample pretreatment including solid phase extraction (SPE) [6] or liquid-liquid extraction [7]. Moreover, they were characterized by incomplete validation protocols which do not follow international guidelines [12,13]. To date no UHPLC-DAD methods have been published on LAmB determination. Our method is based on a rapid-organic protein precipitation that allows us to completely dissolve the lipid formulation of the drug and to quantify the total plasmatic amphotericin B. The extraction procedure has been followed by a chromatographic separation using a 1.9 m UHPLC column which allowed shorter turn around times (8 min) if compared to other published methods [7]. These requirements are necessary for routine TDM analyses. The use of DAD assures the specific detection of LamB by using two different wavelengths. Possible interferences with comedications or other substances have been extensively studied and were absent. The extensive validation protocol applied assures its applicability on routine TDM. 5. Conclusion A robust, rapid and cost-effective UHPLC method was developed and validated following international guidelines for the quantification of LAmB in human plasma. The method can be easily applied for TDM of LAmB on pediatric or adult patients with invasive micoses.
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References [1] N.R. Stone, T. Bicanic, R. Salim, W. Hope, Liposomal amphotericin B ® (AmBisome( )): a review of the pharmacokinetics pharmacodynamics, clinical experience and future directions, Drugs 76 (2016) 485–500. [2] J.P. Botero Aguirre, A.M. Restrepo Hamid, Amphotericin B deoxycholate versus liposomal amphotericin B: effects on kidney function, Cochrane Database Syst. Rev. 23 (2015) CD010481. [3] A.J. Lepak, D.R. Andes, Antifungal pharmacokinetics and pharmacodynamics, Cold Spring Harb. Perspect. Med. 10 (2014) a019653. [4] J.M. Lestner, A.H. Groll, G. Aljayyoussi, N.L. Seibel, A. Shad, C. Gonzalez, L.V. Wood, P.F. Jarosinski, T.J. Walsh, W.W. Hope, Population pharmacokinetics of liposomal amphotericin B in immunocompromised children, Antimicrob. Agents Chemother. (October (3)) (2016), pii: AAC. 01427–16. [Epub ahead of print]. [5] J.W. Mouton, U. Theuretzbacher, W.A. Craig, P.M. Tulkens, H. Derendorf, O. Cars, Tissue concentrations: do we ever learn, J. Antimicrob. Chemother. 61 (2008) 235–237. [6] T. Eldem, N. Arican-Cellat, Determination of amphotericin B in human plasma using solid-phase extraction and high-performance liquid chromatography, J. Pharm. Biomed. Anal. 25 (2001) 53–64. [7] M.P. Lambros, S.A. Abbas, D.W. Bourne, New high-performance liquid chromatographic method for amphotericin B analysis using an internal standard, J. Chromatogr. B Biomed. Appl. 685 (1996) 135–140. [8] R. Lopez-Galera, L. Pou-Clave, C. Pascual-Mostaza, Determination of amphotericin B in human serum by liquid chromatography, J. Chromatogr. B Biomed. Appl. 674 (1995) 298–300. [9] N.M. Deshpande, M.G. Gangrade, M.B. Kekare, V.V. Vaidya, Determination of free and liposomal amphotericin B in human plasma by liquid chromatography-mass spectroscopy with solid phase extraction and protein precipitation techniques, J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 878 (2010) 315–326. [10] W. Qin, H. Tao, Y. Chen, Z. Chen, Wu N. Sensitive, accurate and simple liquid chromatography-tandem mass spectrometric method for the quantitation of amphotericin B in human or minipig plasma, J. Chromatogr. Sci. 50 (2012) 636–643. [11] B.T. Al-Quadeib, M.A. Radwan, L. Siller, E. Mutch, B. Horrocks, M. Wright, A. Alshaer, Therapeutic monitoring of amphotericin B in Saudi ICU patients using UPLC MS/MS assay, Biomed. Chromatogr. 28 (2014) 1652–1659. [12] EMEA/CHMP/EWP/192217/2009, Guideline on Bioanalytical Method Validation, The European Medicines Agency, 2011. [13] Guidance for industry: Bioanalytical Method Validation, US Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (CDER), Center for Biologics Evaluation and Research (CBER), Rockville, USA, 2001.
Contents lists available at ScienceDirect
Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba
A rapid and robust UHPLC-DAD method for the quantification of amphotericin B in human plasma Sebastiano Barco a , Alessia Zunino a , Antonio D’Avolio b , Laura Barbagallo a , Angelo Maffia a , Gino Tripodi a , Elio Castagnola c , Giuliana Cangemi a,∗ a
Clinical Pathology Laboratory Unit, Istituto Giannina Gaslini, Genoa, Italy Department of Medical Sciences, University of Turin, Laboratory of Clinical Pharmacology and Pharmacogenetics, Amedeo di Savoia Hospital, Turin, Italy c Infectious Disease Unit, Istituto Giannina Gaslini, Genoa, Italy b
a r t i c l e
i n f o
Article history: Received 7 December 2016 Received in revised form 23 January 2017 Accepted 24 January 2017 Available online 8 February 2017 Keywords: UHPLC Amphotericin B
a b s t r a c t Amphotericin B is an antifungal drug widely used in Intensive Care Units. Therapeutic drug monitoring (TDM) of amphotericin B is recommended for the assessment of toxicity surveillance and treatment optimization. In this paper we described the development and validation of a new Ultra High Performance Liquid Chromatography coupled to Diode Array Detection (UHPLC-DAD) method for the quantification of Amphotericin B in 200 L human plasma over a wide range of concentrations (0.125–10 mg/L). The new method has been validated following international guidelines on bioanalytical method validation and showed high selectivity, high accuracy and precision and high process efficiency. The new UHPLC-DAD method that we describe is robust, rapid, cost effective and suitable for application to the routine TDM analyses. © 2017 Published by Elsevier B.V.
1. Introduction Amphotericin B is a polyene antifungal agent with activity against molds and yeasts. The liposomal formulation of amphotericin B (LAmB) is widely used for the treatment or prophylaxis of invasive antifungal disease both in adults and in children, with a significant reduced toxicity respect the initial deoxycholate (DAmB) formulation, even if nephro and hepatotoxicity can still be observed [1,2]. Amphotericin B presents a concentration-dependent activity [3] and could be both fungistatic and fungicidal depending on the concentration of the drug in various body fluids and the susceptibility of specific fungus. Therapeutic drug monitoring (TDM) of LAmB could be recommended in special populations such as immunocompromised patients [4] and might be essential in patients in intensive care. Concentrations of LAmB can be measured using high performance liquid chromatography (HPLC), liquid chromatography–mass spectrometry (LC–MS/MS), or bioas-
Abbreviations: UHPLC, ultra high performance liquid chromatography; DAD, diode array detector. ∗ Corresponding author at: Clinical Pathology Laboratory Unit, Istituto Giannina Gaslini, Via Gerolamo Gaslini 5, 16147 Genova. E-mail address: [email protected] (G. Cangemi). http://dx.doi.org/10.1016/j.jpba.2017.01.048 0731-7085/© 2017 Published by Elsevier B.V.
says. The quantification method has a significant impact on what exactly is being measured (i.e. total amphotericin B, protein-bound drug, liposome-associated drug and freely circulating drug). Therefore, caution is required with the interpretation of drug concentrations. The critical step of each method is the extraction of amphotericin B from the liposome. A complete destruction of the liposome by using organic solvents and the release of the active drug needs to be obtained in order to correctly estimate the total concentration of amphotericin B within the matrix [1,5]. To date no commercially available kits for the measurement of amphotericin B by HPLC are currently available. Several HPLC methods have been described for the quantification of amphotericin B in human plasma [6–8], but they are scarcely applicable to routine LamB TDM because poorly validated and characterized by extensive sample preparation protocols [6] or long run times [7]. More recently LC–MS based methods have been published [9–11]. In this paper we describe a new Ultra High Performance Liquid Chromatography coupled to Diode Array Detection (UHPLC- DAD) method for the determination of total amphotericin B in plasma. The new method has been validated according to international guidelines on bioanalytical method validation [12,13] and has been successfully applied to pediatric patients
S. Barco et al. / Journal of Pharmaceutical and Biomedical Analysis 138 (2017) 142–145
2. Materials and methods
143
simultaneously measured at 384 and 407 nm (amphotericin B) and 304 nm (IS).
2.1. Reagents and chemicals 2.6. HPLC method validation Amphotericin B and natamycin (IS) were purchased from Sigma (Milan, Italy). Acetonitrile was purchased from Carlo Erba Reagenti (Milan, Italy). All solutions were prepared with HPLC grade water obtained from a Milli-Q Plus water purification system. HPLC mobile phases were filtered by using Millipore membrane filters [0.45 m] (Millipore, Vimodrone MI, Italy). 2.2. Human samples Plasma was obtained from peripheral blood collected in sodium heparin-containing tubes by centrifuging at 4000 g for 5 min. Plasma samples were stored at −20 ◦ C until analysed. For method validation purposes, blank samples were obtained from healthy adult volunteers who were not being treated with Amphotericin B. Leftover samples derived from seven pediatric patients (age 2 months-18 years) of Intensive Care Units of G. Gaslini Institute who were being treated with Amphotericin B (Ambisome, LamB Gilead Sciences, Paris, France) for the treatment of invasive micoses were obtained. In order to test selectivity, leftover samples derived from 50 pediatric patients (age 6 months-10 years) of Intensive Care Units of G. Gaslini Institute who were not being treated with LamB but who were treated with the same co-medications were obtained. The Internal Review Board was notified about TDM program, but no formal approval was required since no additional blood sampling was required in order to set up the new method. 2.3. Sample preparation A 200 L aliquot of plasma was protein precipitated with 50 L of ZnSO4 0.1 M and 200 L of acetonitrile after addition of 20 L IS (20 mg/L). After vortexing, samples were centrifuged for 10 min at 13.000 × g at 4 ◦ C and the supernatant was transferred to autosampler vials and 10 L were injected in the UHPLC system. 2.4. Preparation of calibrators and quality control samples Stock solutions of 1 g/L amphotericin B in DMSO and 0.53 g/L IS in methanol, were prepared and stored at −20 ◦ C. Calibrators and quality controls (QC) were obtained by serial dilution of stock solutions with plasma obtained from healthy adult volunteers. A 7-point calibration curve was assessed using amphotericin B concentrations of 0.125–0.25–0.5–1–2.5–5–10 mg/L. QC samples were prepared at the following four concentration levels: 0.125 mg/L (LLOQ), 0.375 mg/L (low QC), 1.5 mg/L (medium QC) and 7.5 mg/L (high QC). 2.5. Chromatographic conditions Chromatography was performed by using an Ultimate 3000 UHPLC system (ThermoScientific, Milan, Italy) equipped with a diode array detector (DAD, Ultimate 300RS, Thermoscientific, Milan, Italy). Analysis was performed on a Hypersil Gold column (50 mm × 2.1 mm; particle size, 1.9 m; Thermoscientific, Milan, Italy). Analytes were separated by gradient elution at 50 ◦ C with mobile phase A consisting in 0.1 M KH2 PO4 in H2 O (adjusted at pH = 3 with H3 PO4 ) and mobile phase B of acetonitrile at a flow-rate of 0.8 mL/min. The percentage of mobile phase B was maintained at 10% for 0.2 min and then programmed to reach 100% in 5 min and then maintained 1.5 min. The column was finally reconditioned at 10% B in 1.5 min for a total run time of 8 min. The absorbances were
A laboratory scheme based on EMA and FDA guidelines on bioanalytical method validation [10,11] was used for assay validation. 2.6.1. Selectivity Selectivity was evaluated as a lack of interference of matrix or other concomitant medications by analyzing six different batches of pooled plasma from healthy volunteers. Moreover 50 samples derived from leftover plasma of patients of Institute G. Gaslini in Intensive Cure Units who were not being treated with amphotericin B but who were treated with several co-medications. Interfering components were considered as absent when the signal was less than 20% of the LLOQ for the analytes and less than 5% for the IS. 2.6.2. Carry- over Carry-over was assessed by injecting blank samples in triplicate after the highest calibration standard. The signal in the blank sample following the high standard should not be greater than 20% of the LLOQ and 5% for the IS. 2.6.3. Accuracy and precision Within-day and between-day precision and accuracy were determined at four concentration levels, using the four level QC samples (LLOQ, low, medium and high QC) and by analyzing each sample six separate times. Inter-day precision was estimated by repeating the procedure three times on three separate days. Accuracy was expressed as the mean relative error (percentage of agreement between calculated and nominal concentration of QCs). Precision was expressed as the coefficient of variation (CV%). The acceptance criteria for within- and between-day precision were ≤15% and for accuracy was 85–115% of the nominal concentrations. 2.6.4. Lower limit of quantification (LLOQ) The LLOQ was determined as the lowest sample concentration that provided measurements with an precision ≤20% and accuracy within 80–120% of the nominal concentration. 2.6.5. Linearity Linearity was evaluated by analyzing calibration standards three times on three separate days in the test range 0.125–10 mg/L. The slope, intercept, and correlation coefficient for each calibration curve were obtained by plotting the peak area ratio of analyte IS vs. the nominal concentration of each calibrator. The acceptance criteria for the amounts of back-calculated standard was ± 15% of the theoretical value (except ± 20% for the lowest standard). 2.6.6. Dilution integrity Dilution integrity was investigated by spiking a pool of plasma with amphotericin B at 18 mg/L. After serial dilution 1:2 and 1:4 with blank matrix, each sample was analysed five times. The acceptance criteria were ± 15% of the theoretical value. 2.6.7. Matrix effects and extraction recoveries Matrix effects and extraction recoveries were assessed at two different levels (corresponding to the QC low and QC high ) analysed in triplicate using 6 different batches of pooled plasma. Matrix effects were assessed by comparing peak area of amphotericin B spiked after extraction from plasma to peak area of pure solutions at the same concentration. Extraction recoveries were determined by comparing peak area of amphotericin B spiked before the extraction procedure to peak area of amphotericin B spiked after protein precipitation. Process efficiency was determined by comparing peak
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S. Barco et al. / Journal of Pharmaceutical and Biomedical Analysis 138 (2017) 142–145
Fig. 1. Chromatograms for amphotericin B. Panel A: IS (304 nm); Panel B: amphotericin B (384 nm); Panel C: amphotericin B (407 nm).
Table 1 Precision and accuracy results. Mean±SD (mg/L)
Precision (CV%)
Accuracy (R.E.%)
Mean±SD (mg/L)
Intra-day LLOQ QC low QC medium QC high
0.125 ± 0.02 0.375 ± 0.06 1.5 ± 0.25 7.5 ± 0.75
Precision (CV%)
Accuracy (R.E.%)
5.3 5.6 6.5 4.8
97.6 94.9 94.6 97.3
Inter-day 2.1 2.1 0.9 1.2
0.44 ± 0.02 1.8 ± 0.10 6.43 ± 0.59 26.28 ± 1.55
96.5 98.5 99.2 99.6
Table 2 Matrix effect and extraction recoveries.
Table 3 Stability results.
QC level
Extraction recoveries
Matrix effect
Processefficiency
QC low QC high
100 102.4
103.2 92.8
129.6 119.2
area of amphotericin B spiked before the extraction procedure to peak area of pure solution. Results were considered acceptable with CV < 15%.
Storage Condition
3. Results 3.1. Method development The extraction protocol based on protein precipitation with ZnSO4 and acetonitrile from 200 L plasma allowed a fast and efficient extraction of LamB. The use of Hypersil Gold 1.9 m column allowed us to obtain an excellent peak resolution with a short chromatographic separa-
QC high a
◦
24 h at 25 C 4 h at 25 ◦ C freeze/thaw 3 times 15 days at −20 ◦ C 15 days at +4 ◦ C extracted 3days at 25 ◦ C a
2.6.8. Stability Stability of extracted or unextracted samples was determined on QC low and QC high analysed in triplicate. Short-term temperature stability was determined by maintaining the samples at room temperature for 6 and 24 h. Long term temperature stability was determined by maintaining the samples at −20 ◦ C for 15 days or at +4 ◦ C for 7 or 15 days respectively. Freeze and thaw stability was determined after three freeze and thaw cycles. Stability of extracted samples was determined by maintaining extracts at room temperature for 72 h. Amphotericin B and IS stock solutions stability was determined by maintaining them at −20 ◦ C for 15 or 30 days.
QC low CV (%)
Recovery
CV (%)a
Recovery
4.4 1.1 2.0 1.0 0.5 3.0
93 99 98 99 97 96
9.0 8.0 3.0 0.5 1.0 2.0
98 98 99 99 98 91
CV calculated from three replicates.
tion (with a total run time of 8 min) thus greatly improving the productivity of the method and decreasing mobile phase usage. Chromatograms obtained for both amphotericin B and its IS are shown in Fig. 1. Retention times are 3.57 and 3.12 min for amphotericin B and its IS respectively. 3.2. Method validation The method was validated following international guidelines and showed excellent selectivity with no interfering peaks at the specified chromatographic conditions. Carry-over was absent. The LLOQ was 0.125 mg/L. The linear regression fit for the calibration curves was achieved. All back-calculated values did not differ from ±15% of the theoretical value. The results of the intra-inter assay precision and accuracy and recoveries were all inside the acceptable ranges (Table 1). The dilution integrity assay gave results always under ±15% of the theoretical value at the two dilution factors tested. Matrix effect, extraction recovery and process efficiency tests gave results inside the acceptable ranges (Table 2). Stability tests performed on QCs
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(Table 3) demonstrated that unprocessed samples were stable for at least 24 h at room temperature and for 15 days at 4 ◦ C and at −20 ◦ C and after three freeze/thaw cycles. Processed samples can be left at room temperature for three days without significant losses. The new UHPLC method has been tested on 9 samples derived from 7 pediatric patients treated with amphotericin B. The results were in the range 0.2-5.9 mg/L. 4. Discussion Several HPLC methods [6–8] have been previously described for the quantification of LAmB in human plasma. HPLC methods generally required time-consuming sample pretreatment including solid phase extraction (SPE) [6] or liquid-liquid extraction [7]. Moreover, they were characterized by incomplete validation protocols which do not follow international guidelines [12,13]. To date no UHPLC-DAD methods have been published on LAmB determination. Our method is based on a rapid-organic protein precipitation that allows us to completely dissolve the lipid formulation of the drug and to quantify the total plasmatic amphotericin B. The extraction procedure has been followed by a chromatographic separation using a 1.9 m UHPLC column which allowed shorter turn around times (8 min) if compared to other published methods [7]. These requirements are necessary for routine TDM analyses. The use of DAD assures the specific detection of LamB by using two different wavelengths. Possible interferences with comedications or other substances have been extensively studied and were absent. The extensive validation protocol applied assures its applicability on routine TDM. 5. Conclusion A robust, rapid and cost-effective UHPLC method was developed and validated following international guidelines for the quantification of LAmB in human plasma. The method can be easily applied for TDM of LAmB on pediatric or adult patients with invasive micoses.
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