Research article Received: 19 May 2014,
Revised: 23 July 2014,
Accepted: 15 August 2014
Published online in Wiley Online Library: 23 October 2014
(wileyonlinelibrary.com) DOI 10.1002/bmc.3328
Development and validation of a bioanalytical method by LC-MS/MS for the quantification of the LAFIS 10 – an antimalarial candidate – and its pharmacokinetics first evaluation J. V. Laureanoa, F. Barretoa, S. Gnoattoa, L. Tassob, T. Dalla Costaa and B. V. de Araujoa,c* ABSTRACT: A rapid and highly sensitive method by LC-MS/MS was developed and validated for the quantification of an antimalarial candidate (LAFIS10) in rat plasma using dexamethasone as internal standard (IS). The chromatographic separation was performed with a Poroshell 120 EC-C18 column. The mobile phase consisted of water (A) and acetonitrile (B), both containing 10 mM of ammonium formate and 0.1% formic acid, delivered in the form of elution gradient. The LAFIS10 was monitored using an electrospray ionization interface operating in the positive mode in multiple reaction monitoring mode, monitoring the transitions 681.47 → 538.2 for LAFIS10 and 393.20 → 355.30 for the IS. The flow rate was 500 μL/min. The column temperature was kept at 40 °C and the injection volume was 2 μL. The lower limit of quantification was of 10 ng/mL and linearity between 10 and 1000 ng/mL was observed, with an R2 > 0.99. The accuracy of the method was >90%. The relative standard deviations intra- and interday were 538.2. (E) A plasma sample processed after 8 h of the administration of 2.5 mg/kg i.v. containing 500 ng/mL of IS and (F) 52 ng/mL of LAFIS10.
Biomed. Chromatogr. 2015; 29: 664–670
Linearity The range of concentration used to validate the standard curve was 10–1000 ng/mL, the range in which linearity of the analytical method was observed. The mean of the seven standard curves in plasma used in the validation of the analytical method by
Copyright © 2014 John Wiley & Sons, Ltd.
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endogenous substances that may interfere with detection of the compounds of interest were observed. The retention times of LAFIS 10 and IS were approximately 2.6 and 2.2 min, respectively. The extraction recovery of LAFIS 10 was 97.6 ± 3.6%, regardless the analyte concentration in the sample. The recovery of the IS was 97.1 ± 4.1%.
J. V. Laureano et al. LC-MS/MS is shown in Fig. 3, with its equation of the straight line and coefficient of determination (R2). The parameters of the standard curve were determined on two consecutive days for the validation and are shown in Table 2. The standard curves (peak area ratio LAFIS 10/IS) against the nominal concentration were linear, showing a higher coefficient of determination or equal to 0.99 for all curves, and this value exceeds the limit set by the US Food and Drug Administration, which is 0.95 for biological samples.
Carry-over No carry-over effect was observed in the evaluation of the chromatograms resulting from the injection of mobile phase (without analyte) after successive injections of the highest concentration of the standard curve (1000 ng/mL).
Matrix effect The matrix factor was determined for six independent batches of plasma. The coefficient of variation for the ratio analyte/IS was
Revised: 23 July 2014,
Accepted: 15 August 2014
Published online in Wiley Online Library: 23 October 2014
(wileyonlinelibrary.com) DOI 10.1002/bmc.3328
Development and validation of a bioanalytical method by LC-MS/MS for the quantification of the LAFIS 10 – an antimalarial candidate – and its pharmacokinetics first evaluation J. V. Laureanoa, F. Barretoa, S. Gnoattoa, L. Tassob, T. Dalla Costaa and B. V. de Araujoa,c* ABSTRACT: A rapid and highly sensitive method by LC-MS/MS was developed and validated for the quantification of an antimalarial candidate (LAFIS10) in rat plasma using dexamethasone as internal standard (IS). The chromatographic separation was performed with a Poroshell 120 EC-C18 column. The mobile phase consisted of water (A) and acetonitrile (B), both containing 10 mM of ammonium formate and 0.1% formic acid, delivered in the form of elution gradient. The LAFIS10 was monitored using an electrospray ionization interface operating in the positive mode in multiple reaction monitoring mode, monitoring the transitions 681.47 → 538.2 for LAFIS10 and 393.20 → 355.30 for the IS. The flow rate was 500 μL/min. The column temperature was kept at 40 °C and the injection volume was 2 μL. The lower limit of quantification was of 10 ng/mL and linearity between 10 and 1000 ng/mL was observed, with an R2 > 0.99. The accuracy of the method was >90%. The relative standard deviations intra- and interday were 538.2. (E) A plasma sample processed after 8 h of the administration of 2.5 mg/kg i.v. containing 500 ng/mL of IS and (F) 52 ng/mL of LAFIS10.
Biomed. Chromatogr. 2015; 29: 664–670
Linearity The range of concentration used to validate the standard curve was 10–1000 ng/mL, the range in which linearity of the analytical method was observed. The mean of the seven standard curves in plasma used in the validation of the analytical method by
Copyright © 2014 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/bmc
667
endogenous substances that may interfere with detection of the compounds of interest were observed. The retention times of LAFIS 10 and IS were approximately 2.6 and 2.2 min, respectively. The extraction recovery of LAFIS 10 was 97.6 ± 3.6%, regardless the analyte concentration in the sample. The recovery of the IS was 97.1 ± 4.1%.
J. V. Laureano et al. LC-MS/MS is shown in Fig. 3, with its equation of the straight line and coefficient of determination (R2). The parameters of the standard curve were determined on two consecutive days for the validation and are shown in Table 2. The standard curves (peak area ratio LAFIS 10/IS) against the nominal concentration were linear, showing a higher coefficient of determination or equal to 0.99 for all curves, and this value exceeds the limit set by the US Food and Drug Administration, which is 0.95 for biological samples.
Carry-over No carry-over effect was observed in the evaluation of the chromatograms resulting from the injection of mobile phase (without analyte) after successive injections of the highest concentration of the standard curve (1000 ng/mL).
Matrix effect The matrix factor was determined for six independent batches of plasma. The coefficient of variation for the ratio analyte/IS was
