Development and Validation of a Simple HPLC-UV Assay Method for Determination of Levetiracetam Concentrations in Human Plasma
Development and Validation of a Simple HPLC-UV Assay Method for Determination of Levetiracetam...
Kharouba, Maged;Mahmoud, Sherif Hanafy
Article Development and Validation of a Simple HPLC-UV Assay Method for Determination of Levetiracetam Concentrations in Human Plasma Maged Kharouba and Sherif Hanafy Mahmoud * Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, AB T6G 2E1, Canada * Correspondence: email@example.com; Tel.: +1-780-492-5364 Abstract: Levetiracetam (LEV) is a broad spectrum antiseizure medication that is used in various seizure types. There is evidence that therapeutic drug monitoring (TDM) of LEV is of value in selected patient populations, therefore determination of LEV plasma concentrations is essential. Herein we developed and validated a simple, reproducible, and practical method for the quantiﬁcation of LEV concentrations in human plasma samples using high performance liquid chromatography (HPLC). Plasma samples (0.3 mL) deproteinization was done using acetonitrile. HPLC chromatographic separation of plasma samples was accomplished by reversed phase C18 column. The mobile phase constituted water and acetonitrile (90:10, v/v) ran at ﬂow rate of 1 mL/min. Signal acquisition was conducted at a wavelength of 192 nm. Calibration curves showed excellent linearity (Correlation coefﬁcient r > 0.99) over a concentration range of 3–80 g/mL. Both inter and intraday assay accuracy and precision were less than 8% (except for the lowest limit of quantiﬁcation was within 20%). Elution time was 15 min. The developed method excluded the use of buffers and utilized small volumes of plasma samples with simple mobile phase composition. Therefore, our method could be practically applied to routine TDM. Keywords: levetiracetam; HPLC; therapeutic drug monitoring; antiseizure medications; chromatog- raphy; epilepsy Citation: Kharouba, M.; Mahmoud, S.H. Development and Validation of 1. Introduction a Simple HPLC-UV Assay Method for Determination of Levetiracetam Levetiracetam (LEV), (-)-(S)--ethyl-2-oxo-1-pyrrolidine acetamide, is a broad spec- Concentrations in Human Plasma. trum antiseizure medication (ASM) that is used in various types of seizures, either as Analytica 2023, 4, 1–9. https:// monotherapy or in combination with other ASMs (Figure 1) . LEV has a more plau- doi.org/10.3390/analytica4010001 sible adverse effect proﬁle and less propensity for drug interactions compared to older ASMs such as phenytoin and valproic acid. As a result, LEV is now in the forefront in Academic Editor: Marcello Locatelli epilepsy management across the age spectrum. LEV exhibits a linear pharmacokinetic Received: 12 November 2022 proﬁle, rapidly absorbed with relatively complete absorption after oral administration, ap- Revised: 19 December 2022 parently low plasma protein binding (<10%), and is renally eliminated (66% as unchanged Accepted: 29 December 2022 drug) and metabolized by non-hepatic enzymatic hydrolysis . Additionally, there are no Published: 4 January 2023 signiﬁcant interactions between LEV and the other ASMs since LEV disposition is inde- pendent on the human liver cytochrome P450 enzymes . Although LEV has predictable pharmacokinetics, LEV therapeutic drug monitoring (TDM) has been suggested in certain patient populations owing to LEV pharmacokinetic alterations, particularly in patients Copyright: © 2023 by the authors. with renal impairment, children, older adults, pregnant women and those who are critically Licensee MDPI, Basel, Switzerland. ill [4,5]. The suggested reference plasma concentration range for LEV has been reported This article is an open access article to range from 12 to 46 g/mL . Accordingly, quantiﬁcation of LEV plasma concentra- distributed under the terms and tions is crucial for TDM. Herein, we developed a reproducible, simple, and sensitive high conditions of the Creative Commons performance liquid chromatography with ultra-violet detection (HPLC-UV) method for Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ LEV quantiﬁcation in human plasma samples, enabling this analytical method suitable 4.0/). for pharmacokinetic and research studies. Our method employed a single plasma protein Analytica 2023, 4, 1–9. https://doi.org/10.3390/analytica4010001 https://www.mdpi.com/journal/analytica Analytica 2023, 4, FOR PEER REVIEW 2 Analytica 2023, 4 2 for LEV quantification in human plasma samples, enabling this analytical method suitable for pharmacokinetic and research studies. Our method employed a single plasma protein precipitation step, utilized small plasma sample volume (0.3 mL) as compared to 0.5–1 precipitation step, utilized small plasma sample volume (0.3 mL) as compared to 0.5–1 mL mL sample volume in most methods [6–10], and used a simple water/acetonitrile mixture sample volume in most methods [6–10], and used a simple water/acetonitrile mixture for mobile phase, compared to buffers in the majority of methods [6–8,10–16]. Moreover, for mobile phase, compared to buffers in the majority of methods [6–8,10–16]. Moreover, our developed method quantifies LEV throughout a range of 3–80 g μ /mL that covers the our developed method quantiﬁes LEV throughout a range of 3–80 g/mL that covers the suggested reference range (12–46 μg/mL) for LEV TDM. suggested reference range (12–46 g/mL) for LEV TDM. Figure 1. Levetiracetam chemical structure. Figure 1. Levetiracetam chemical structure. 2. Materials and Methods 2. Materials and Methods 2.1. Chemicals and Reagents 2.1. Chemicals and Reagents Levetiracetam (purity > 98%) and caffeine (purity 99%), internal standard (IS), were Levetiracetam (purity > 98%) and caffeine (purity ≥ 99%), internal standard (IS), were obtained from Fisher Scientiﬁc Company (Ottawa, ON, Canada), HPLC-grade acetonitrile obtained from Fisher Scientific Company (Ottawa, ON, Canada), HPLC-grade acetonitrile and HPLC-grade water were obtained from Sigma-Aldrich (Oakville, ON, Canada), and and HPLC-grade water were obtained from Sigma-Aldrich (Oakville, ON, Canada), and blank human plasma was obtained from Cedarlane Laboratories (Burlington, ON, Canada). blank human plasma was obtained from Cedarlane Laboratories (Burlington, ON, Can- 2.2. Instrument ada). The experiment was conducted using a Shimadzu HPLC system (Shimadzu, Kyoto, 2. Japan) 2. Instr consisting ument of two solvent delivery pumps (LC-10 ADvp), SIL-HTc autosampler and system controller coupled to a UV-Vis detector (SPD-10 AV). The HPLC chromatographic The experiment was conducted using a Shimadzu HPLC system (Shimadzu, Kyoto, separation was performed using a reverse phase Supleco Discovery C18 column (5 m, Japan) consisting of two solvent delivery pumps (LC-10 ADvp), SIL-HTc autosampler and 250 4.6 mm) (Supleco Inc., Mississauga, ON, Canada) with a Discovery C18 Supel- system controller coupled to a UV-Vis detector (SPD-10 AV). The HPLC chromatographic guard™ guard column (5 m, 20 4 mm) (Supleco Inc., Mississauga, ON, Canada). separation was performed using a reverse phase Supleco Discovery C18 column (5 μm, Clarity software version 8.7 (DataApex, Prague, The Czech Republic) was used for acqui- 250 × 4.6 mm) (Supleco Inc., Mississauga, ON, Canada) with a Discovery C18 Supel- sition of data and analysis integration. guard™ guard column (5 m μ , 20 × 4 mm) (Supleco Inc ., Mississauga, ON, Canada). Clar- ity software version 8.7 (DataApex, Prague, The Czech Republic) was used for acquisi- 2.3. Chromatographic Conditions tion of data and analysis integration. A mobile phase mixture of water and acetonitrile (90:10, v/v) and an isocratic elution with a ﬂow rate set to 1 mL/min was used for eluting LEV and caffeine (IS). The total run 2.3. Chromatographic Conditions time for elution was 15 min. The wavelength for detection was 192 nm. The column and A mobile phase mixture of water and acetonitrile (90:10, v/v) and an isocratic elution the autosampler were operating at room temperature. with a flow rate set to 1 mL/min was used for eluting LEV and caffeine (IS). The total run time for elution was 15 min. The wavelength for detection was 192 nm. The column and 2.4. Preparation of Standard and Working Solutions the autosampler were operating at room temperature. LEV and caffeine were dissolved in water to prepare a 1 mg/mL standard stock solu- tions. Working solutions of 100 g/mL and 350 g/mL of LEV and caffeine, respectively, 2.4. Preparation of Standard and Working Solutions were produced by further diluting the stock solutions with HPLC grade water. The stock LEV and caffeine were dissolved in water to prepare a 1 mg/mL standard stock solu- and working solutions were freshly prepared daily. tions. Working solutions of 100 g μ /mL and 350 g μ /mL of LEV and caffeine, respecti vely, 2.5. Preparation of Calibration Concentrations and Quality Control (QC) Samples were produced by further diluting the stock solutions with HPLC grade water. The stock and working solutions were freshly prepared daily. We prepared serial dilutions of LEV concentrations in blank plasma samples to gen- erate calibration curves. LEV concentrations ranged from 3 to 80 g/mL. We prepared 4 QC samples for method validation: low limit of quantiﬁcation sample (LLOQ), low Analytica 2023, 4 3 level QC sample (LQC), middle level QC sample (MQC; within the middle range of calibration concentrations) and high level QC sample (HQC; close to the upper end of calibration concentrations). 2.6. Sample Preparation A total of 300 L of plasma samples were mixed with 50 L of 350 g/mL caffeine (IS). Plasma samples were vortexed for 1 min. Then, 3 mL of acetonitrile were added to the samples for protein precipitation followed by vortex mixing for 5 min. Samples were then centrifuged at 4000 rpm for 20 min at 4 C. The supernatant was then transferred to clean tubes and evaporated using a SpeedVac Vacuum Concentrator (Thermo Fisher Scientiﬁc, Waltham, MA, USA). Reconstitution of the residue was made using a 350 L mobile phase (water: acetonitrile, 90:10, v/v) and vortex mixed for 10 s. A 40 L of each sample was then injected into the HPLC. 2.7. Method Validation The developed method was validated according to the guidelines on bioanalytical method validation by the European Medicines Agency (EMA, 2011) . We evaluated the method’s linearity, selectivity, sensitivity, precision, accuracy, carry-over effect, dilution integrity, stability and extraction recovery. 2.7.1. Linearity For method’s linearity assessment, calibration curves were constructed by plotting the peak area ratios (LEV/internal standard) vs. the concentrations of the calibration standards (3, 10, 20, 40, 60, and 80 g/mL). Linear regression was utilized to determine the calibration curve parameters: the slope, intercept, and the correlation coefﬁcient (r ). 2.7.2. Selectivity and Sensitivity We conﬁrmed method selectivity by the absence of any peaks at the analyte and IS retention times when we injected blank plasma samples. The LLOQ is the lowest concentration within the calibration curve that has a precision < 20%, accuracy within 20% and its signal is at least 5 times higher than that of blank plasma. 2.7.3. Precision and Accuracy We determined the precision and accuracy of our method by injecting ﬁve replicates of QC samples that were spiked with LEV at four different QC levels (3, 30, 50 and 70 g/mL), conducted over three consecutive days (3 separate runs). The method’s precision was expressed as coefﬁcient of variation (CV, %), whereas the accuracy was described as percentage error. We then determined the intra and interday precision and accuracy of the method. 2.7.4. Carry-Over Carry-over was evaluated by injecting human blank plasma following the ULOQ. EMA guidelines acceptability criterion for carry-over was that the carry-over in the injected blank sample preceded by the ULOQ should not exceed 5% of the IS signal and 20% of LLOQ signal. 2.7.5. Dilution Integrity To investigate method’s dilution integrity, we diluted a spiked plasma sample using blank plasma sample matrix with a 15-fold concentration. The accuracy and precision should be within 15% for an acceptable dilution integrity. Analytica 2023, 4 4 2.7.6. Stability We determined the stability of samples by comparing the results of freshly prepared samples (60 g/mL, n = 5) analyzed prior and following exposure to different conditions. The stability conditions mimicked similar conditions for sample handling, storage and analysis. We assessed the stability of LEV in human plasma at room temperature (for 8 h and for 30 days), and at 80 C for 30 days. We also assessed 3 cycles of freeze–thaw sample stability. Each cycle involved freezing for 24 h followed by thawing at room temperature and then the cycle gets repeated. Storage stability of the stock solution was also evaluated at 4 and 80 C. A stability to reference samples ratio within 85 and 115% and a percent error within 15% were considered acceptable. Moreover, reinjection stability was also assessed 24 h after the initial injection. 2.7.7. Recovery The developed method’s average extraction recovery of LEV was assessed by compar- ing the peak areas of the 5 replicates of extracted samples at the 3 QC levels with the peak areas obtained from extracted human blank plasma (same extraction procedures) that were spiked with equivalent concentrations of LEV after extraction. 3. Results 3.1. Optimization of Chromatographic Conditions To optimize our method, we evaluated the efﬁciency of two extracting solvents, differ- ent solvent volumes, various detection wavelengths, and diverse mobile phase composi- tions. To improve the separation of LEV peak, the following mobile phase compositions were tested: 90:10, 70:30, 50:50, 40:60, 20:80, and 10:90, v/v acetonitrile:water. The mo- bile phase composition of 10:90 acetonitrile:water v/v showed the best separation and peak intensity. The resultant retention times for LEV and IS were 7.8 and 13.2 min, re- spectively (Figure 2). Nonetheless, plasma non-interfering peaks were also present in the chromatogram. However, excellent linearity calibration curves were achieved. To further enhance peak intensity, we evaluated 5 different detection wavelengths: 190, 192, 195, 200, and 205 nm. Among them, the 192 nm gave the highest peak intensity for LEV plasma samples. Regarding sample extraction, two solvents and 2 different volumes were tested, acetonitrile and methanol, either 1.5 or 3 mL. Acetonitrile 3 mL yielded superior extraction efﬁciency. 3.2. Method Validation 3.2.1. Linearity Assessment of method linearity was performed by running extracted and pure samples throughout LEV calibration concentration ranges. The ratios of peak areas of LEV/IS were linear over the calibration ranges (3–80 g/mL) with correlation factor (r ) > 0.99 (Figure 3). 3.2.2. Selectivity and Sensitivity As displayed in Figure 2, no interfering peaks were present for both LEV and caffeine at their retention times. The LLOQ of LEV was found to be 3 g/mL with intra- and inter-day accuracy and precision were within 20%. 3.2.3. Precision and Accuracy To assess the method’s interday and intraday performance, we analyzed ﬁve replicates of QC samples that were spiked with LEV at four different QC levels. As depicted in Table 1, acceptable CV and percentage error for both intraday and interday were fulﬁlled. The intraday CV and percentage error were within 11.5% and 5%, respectively. Interday CV and percentage error was within 9% and 5%, respectively. Interday percent error was within 20% for the LLOQ. Analytica 2023, 4 5 Analytica 2023, 4, FOR PEER REVIEW 5 (A) (B) Figure 2. (A) A blank plasma chromatogram; (B) a chromatogram showing a sample of human Figure 2. (A) A blank plasma chromatogram; (B) a chromatogram showing a sample of human plasma spiked with 80 μg/mL levetiracetam (LEV) and caffeine as internal standard (IS). LEV and plasma spiked with 80 g/mL levetiracetam (LEV) and caffeine as internal standard (IS). LEV and IS IS retention times were 7.8 and 13.2 min, respectively. retention times were 7.8 and 13.2 min, respectively. 3.2. Method Validation 3.2.1. Linearity Assessment of method linearity was performed by running extracted and pure sam- ples throughout LEV calibration concentration ranges. The ratios of peak areas of LEV/IS were linear over the calibration ranges (3–80 μg/mL) with correlation factor (r ) > 0.99 (Figure 3). Analytica 2023, 4 6 Analytica 2023, 4, FOR PEER REVIEW 6 Calibration curve R² = 0.9931 1.8 1.6 1.4 1.2 0.8 0.6 0.4 0.2 0 10 20 30 40 50 60 70 80 90 LEV plasma concentration (µg/mL) Figure 3. A representative calibration curve of Levetiracetam throughout the range 3–80 μg/mL. Figure 3. A representative calibration curve of Levetiracetam throughout the range 3–80 g/mL. 3.2.2. Selectivity and Sensitivity Table 1. Method inter- and intra-day precision (%CV) and accuracy (% error) for Levetiracetam. As displayed in Figure 2, no interfering peaks were present for both LEV and caffeine QC Concentration Observed Concentration at their retention times. The LLOQ of LEV was found to be 3 μg/mL with intra - and inter- Precision, CV (%) Accuracy, Error (%) (g/mL) (g/mL) Mean SD day accuracy and precision were within ±20%. 3 3.14 0.36 11.35 4.74 30 29.02 0.84 2.88 3.28 3.2.3. Precision and Accuracy Intraday 50 47.76 3.2 6.72 4.48 To assess the method`s interday and intraday performance, we analyzed five repli- 70 69.82 2.67 3.82 0.25 cates of QC samples that were spiked with LEV at four different QC levels. As depicted 3 3.47 0.3 8.77 15.78 in Table 1, acceptable CV and percentage error for both intraday and interday were ful- 30 31.28 2.3 7.38 4.27 Interday filled. The intraday CV and percentage error were within 11.5% and ±5%, respectively. 50 48.69 1.99 4.08 2.62 70 67.92 1.66 2.45 2.96 Interday CV and percentage error was within 9% and ± 5%, respectively. Interday percent error was within ±20% for the LLOQ. Intraday (n = 5 per concentration); Interday (n = 15 per measured concentration over 3 days); CV, coefﬁcient of variation, QC, quality control. Table 1. Method inter- and intra-day precision (%CV) and accuracy (% error) for Levetiracetam. 3.2.4. Carryover QC Concentration Observed Concentration (μg/mL) Mean ± Precision, CV Accuracy, Concerning carryover assessment, no interfering peaks with areas > 20% of the peak (μg/mL) SD (%) Error (%) areas of the LLOQ of LEV were detected in blank samples that were injected post the 3 3.14 ± 0.36 11.35 −4.74 ULOQ sample. 30 29.02 ± 0.84 2.88 3.28 Intraday 3.2.5. Dilution integrity 50 47.76 ± 3.2 6.72 4.48 70 69.82 ± 2.67 3.82 0.25 Assessment of dilution integrity results, in terms of precision and accuracy, revealed that dilution of samples with blank human plasma had no effect on accurately determining 3 3.47 ± 0.3 8.77 −15.78 LEV concentrations higher than the ULOQ. 30 31.28 ± 2.3 7.38 −4.27 Interday 50 48.69 ± 1.99 4.08 2.62 3.2.6. Stability 70 67.92 ± 1.66 2.45 2.96 Stability results are depicted in Table 2. LEV stock solutions were observed to be Intraday (n = 5 per concentration); Interday (n = 15 per measured concentration over 3 days); CV, stable for at least 2 months at 80 C, suggesting that new stock solutions can be made in coefficient of variation, QC, quality control. 2 months interval. Moreover, room temperature stability showed that plasma LEV samples were stable at room temperature for up to 12 h with precision (CV%) and accuracy within 3.2.4. Carryover 15%, assuring that there was insigniﬁcant breakdown of LEV for up to 12 h. Post three Concerning carryover assessment, no interfering peaks with areas > 20% of the peak freeze–thaw cycles, precision and accuracy of LEV concentrations were also 15% from areas of the LLOQ of LEV were detected in blank samples that were injected post the nominal concentrations. Furthermore, LEV extracted plasma samples were stable for at ULOQ sample. least 1 month at 80 C or room temperature. Reinjection stability showed acceptable precision and accuracy (15%) after 24 h. LEV/IS peak ratio Analytica 2023, 4 7 Table 2. Levetiracetam stability testing. Mean Calculated Stability Test Concentration (g/mL) CV (%) % Error Concentrations (g/mL) SD Stock solution stability at 80 C (1 month) 60 60.06 6.53 10.87 0.11 Stock solution stability at 80 C (2 month) 60 63.63 0.24 0.38 6.06 Stock solution stability at 4 C (1 month) 60 63.63 2.61 4.1 6.03 Long term stability at room temperature (1 month) 60 60.92 2.81 3.95 1.5 Long term stability at 80 C (1 month) 60 66.75 1.35 2.02 11.26 Free-thaw stability 60 64.87 1.16 1.79 8.12 Bench-top stability (12 h) 60 66.51 0.79 1.19 10.85 Reinjection stability 60 52.02 0.64 1.49 13.28 n = 5 per concentration; CV, coefﬁcient of variation; IS, internal standard. 3.2.7. Recovery By analyzing the 5 replicates of three QC concentrations for LEV (30, 50, 70 g/mL), the average extraction recovery obtained is displayed in Table 3. LEV average percentage recovery was from 76.75 1.63 % to 80.38 6.43 with a CV of <8%. Table 3. Mean percent recovery for Levetiracetam and Caffeine (IS). Drug Concentration (g/mL) Recovery, Mean SD (%) CV (%) 30 80.38 6.43 8.0 50 77.36 1.60 2.06 Levetiracetam 70 76.75 1.64 2.13 Caffeine (IS) 50 83.39 1.8 2.17 n = 5 per concentration; CV, coefﬁcient of variation; IS, internal standard. 4. Discussion Levetiracetam, marketed since 1999 , is an ASM that has been shown to be effective in various types of seizures. In the current study, we presented a simple, reproducible, and selective method for quantitively measuring LEV in human plasma samples. Analysis of plasma samples can be performed over 15 min, which is comparable or faster than the previously reported methods. We chose caffeine as an internal standard because it has superior extraction recovery and no interference due to limited use in ICU patients. Drug analysis in biological samples as human plasma requires sample pretreatment to remove interfering components and proteins before analysis. Preliminary tests to choose the appropriate deproteinizing solvent and its volume were performed. Methanol and acetonitrile were examined. Acetonitrile was chosen as the deproteinizing solvent as it obtained higher % recovery results. The volume of 3 mL results in superior extraction efﬁciency when compared to 1.5 mL of deproteinizing solvent. Our method requires a single protein precipitation step, and only single isocratic pump and detection channel for our tested analyte, with sufﬁcient extraction recovery (>76%). Moreover, an essential advantage of the developed method when compared to the majority of methods reported is the apparently smaller volume of plasma samples needed (0.3 mL as opposed to 0.5–1 mL in most methods) [6–10], making this chromatographic method feasible for routine monitoring of LEV in human plasma. Additionally, we utilized only a simple isocratic mixture of water and acetonitrile without any buffers (as opposed to majority of reported methods) and that have added to the simplicity of our method and with no effect on the performance of separation [6–8,10–16]. Using buffers can have many pitfalls as affecting the column efﬁciency with routine use, adding to the complexity and cost of the method, and is time-consuming, owing to the time needed for extensive column wash after analysis. Nonetheless, stability of our method was assessed, and LEV was found to be stable at least 1 month either in stock solution or in human plasma at Analytica 2023, 4 8 room temperature, refrigerator and at 80 C, assuring safe handling, storage and analysis of LEV using our validated method. The proposed method also showed no signiﬁcant carryover effect. Additionally, the reported reference concentration range for LEV for seizure control ranges between 12–46 mg/mL, and that falls within our validated range (3–80 mg/mL). Therefore, our developed method is feasible in different research activities and also routine LEV TDM. 5. Conclusions In this study, we presented a fully validated chromatographic HPLC-UV method for the quantiﬁcation of LEV in human plasma samples. The developed method was simple, reproducible, and sensitive. It was simple as we used a single mobile phase composition with no buffers and with a single extraction step. The developed method was also suitable to quantify LEV concentration ranges that cover the suggested LEV reference range used in clinical practice. This is in addition to the small sample volume required for analysis. Collectively, the simplicity of the method cuts the cost and time of analysis, making it more practical for day-to-day TDM practice and for research purposes. Author Contributions: Conceptualization, S.H.M.; methodology, S.H.M. and M.K.; validation, S.H.M. and M.K.; formal analysis, S.H.M. and M.K.; writing—original draft preparation, M.K.; writing— review and editing, S.H.M. and M.K.; supervision, S.H.M.; funding acquisition, S.H.M. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the University Hospital Foundation (UHF), Edmonton, Alberta, Canada and the Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta, Canada. Data Availability Statement: All relevant data are within the manuscript. Acknowledgments: Fadumo Ahmed Isse and Fatma Hefny for their assistance with laboratory skills training. Conﬂicts of Interest: The authors declare no conﬂict of interest. References 1. Patsalos, P. The pharmacokinetic characteristics of levetiracetam. Methods Find Exp. Clin. Pharmacol. 2003, 25, 123–129. [CrossRef] [PubMed] 2. Patsalos, P. 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