Research article Received: 18 August 2014,
Revised: 7 January 2015,
Accepted: 16 February 2015
Published online in Wiley Online Library: 24 March 2015
(wileyonlinelibrary.com) DOI 10.1002/bmc.3458
Determination of corilagin in rat plasma using a liquid chromatography–electrospray ionization tandem mass spectrometric method Qian Qian Chena, Jianru Guoa, Hongyan Fanb, Caiyun Wanga, Fengguo Xub and Wei Zhanga* ABSTRACT: A sensitive and simple liquid chromatography–tandem mass spectrometric (HPLC-MS/MS) method for the determination of corilagin in rat plasma has been developed. Samples were prepared with protein precipitation method and analyzed with a triple quadrupole tandem mass spectrometer. We employed negative electrospray ionization as the ionization source and the analytes were detected in multiple reaction monitoring mode. Separation was achieved on a C8 column eluted with mobile phase consisting of methanol–0.1% formic acid in a gradient mode at the flow rate of 0.3 mL/min. The total run time was 7.0 min.This method was proved to have good linearity in the concentration range of 2.5–1000.0 ng/mL. The lower limit of quantification of corilagin was 2.5 ng/mL. The intra- and inter-day relative standard deviationa across three validation runs for four concentration levels were both 5 days. The rat was fasted for 12 h with free access to water before the experiment. All animal experimental procedures in this study were carried out following the guidelines for the care and use of laboratory animals. The rat was orally administered the crude ethanol extract of Phyllanthus urinaria L. (1.0 g/kg) by gavage. Blood samples (approximately 0.3 mL) were collected from the suborbital vein into heparinized centrifuge tubes at 0.0, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0
wileyonlinelibrary.com/journal/bmc
Copyright © 2015 John Wiley & Sons, Ltd.
Biomed. Chromatogr. 2015; 29: 1553–1558
Corilagin Rat Plasma LC-MS/MS and 8.0 h after oral administration. Plasma was separated by centrifugation at 5000 rpm for 10 min and then stored at 80°C until analysis.
in set 2 with that of the standards in set 1. The extraction recoveries were determined by calculating the ratios of the peak areas of the regularly prepared plasma samples in set 3 with that of the spiked post-extraction samples in set 2.
Method validation The current HPLC-MS/MS method was completely validated in biological samples for specificity, linearity, lower limit of quantification (LLOQ), accuracy, precision, extraction recovery, matrix effect (ME) and stability according to the US Food and Drug Administration guidance for bioanalytical method validation. The specificity was evaluated by comparing the chromatograms of the blank rat plasma from six individual rats with the corresponding plasma spiked with corilagin and IS and the plasma sample obtained 4 h after oral administration of Phyllanthus urinaria L. extraction. Plasma samples were quantified using the ratio of the peak area of analyte to that of IS as the assay parameter. Peak area ratios were plotted against analyte concentrations and standard curves were in the form of ‘y = a + bx’. To evaluate linearity, plasma calibration curves were prepared and detected in duplicate on three separate days. The accuracy and precision were also evaluated by determining QC samples at four concentration levels on three different validation days. The accuracy was calculated by (mean observed concentration theoretical concentration)/(theoretical concentration) × 100% and the precision by relative standard deviation (RSD%). Corilagin stability in rat plasma was assessed by analyzing QC samples at concentrations of 5, 50, 250 and 750 ng/mL, respectively, exposed to different time and temperature conditions. The long-term stability was assessed after storage of the test samples at 80°C for 12 days. The freeze–thaw stability was determined after three freeze–thaw cycles, in which the QC samples were thawed completely at room temperature and then refrozen at 80°C for at last 12 h. Autosampler stability was tested by leaving all processed samples in the autosampler at 4°C for 24 h before injection. Matrix effects and extraction recoveries of corilagin were validated in quintuple by comparing the peak areas of the analytes between four different sets of standards of QC concentrations (5, 50, 250 and 750 ng/mL). The first set (set 1) was prepared in 50% methanol. The second set (set 2) was spiked in drug-free plasma extracts and third set (set 3) was prepared in drug-free plasma carrying extracted following ‘Sample preparation’. Matrix effect was assessed by comparing the peak areas of the analyte
Results and discussion Mass spectrometry Negative ionization mode was initially chosen owing to numerous hydroxyl groups (Fig. 1) in both corilagin and rutin. The Q1 full-scan spectra of corilagin and IS dominated by protonated molecules [M H] was significantly observed. When the CID energy was set at 43 and 37 V, the main fragment ion at m/z 633.0–301.0 from corilagin and m/z 609.0–301.0 from rutin showed the highest MS response (Fig. 2). [email protected] Chromatography To decrease the interference of endogenous substances in plasma, a small amount of formic acid was added into the mobile phase. Theoretically, formic acid could improve the resolution of chromatography since most endogenous interference is of an acidic nature but it will in response suppress the MS signal of target compound. Concentrations of 0.01, 0.05, 0.1 and 0.15% formic acid were tested for separation. Finally, 0.1% formic acid was selected because of its high separation efficiency and good MS response. Under these optimum chromatographic conditions, the symmetry of the peak was relative good and the analyte and internal standard were free of interference from endogenous substances. Preparation of plasma samples Protein precipitation is the most widely used biological sample preparation method. In this experiment, methanol, acetonitrile and trichloroacetic acid were all tested for extraction and methanol was finally adopted since it assured high analysis recoveries and stability for the analyzed samples. Method validation Specificity. The specificity tested the ability of the method to differentiate and quantitate the analyte in the presence of other endogenous constituents in the sample and to detect potential
Biomed. Chromatogr. 2015; 29: 1553–1558
Copyright © 2015 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/bmc
1555
Figure 2. Negative ion electrospray mass scan spectra of corilagin (A) and rutin (B).
Q. Q. Chen et al. interferences. The chromatographs of rat blank plasma, the same plasma spiked at the LLOQ of corigagin and the plasma sample obtained at 4 h from a rat after oral administration of Phyllanthus urinaria L. extract are shown in Fig. 3. There was no significant interference or ion suppression from endogenous substances observed at the retention times of the analytes. Typical retention times for corilagin and IS were about 2.42 and 2.68 min, respectively. Linearity of calibration curves and lower limits of quantification. Standard curves were performed in triplicate for each analyte in plasma. In all cases the regression coefficient was >0.99. Corilagin curves showed good linearity over a range of 2.5–1000 ng/mL with a weighting on 1/x2. Typical standard curves were f = 0.0132Ci 0.0118 for corilagin, where f represents the ratio of analyte peak area to that of IS and Ci is the plasma concentrations of analyte. The LLOQ was defined as the lowest concentration on the calibration curve for which an acceptable accuracy of ±15% and a precision
Revised: 7 January 2015,
Accepted: 16 February 2015
Published online in Wiley Online Library: 24 March 2015
(wileyonlinelibrary.com) DOI 10.1002/bmc.3458
Determination of corilagin in rat plasma using a liquid chromatography–electrospray ionization tandem mass spectrometric method Qian Qian Chena, Jianru Guoa, Hongyan Fanb, Caiyun Wanga, Fengguo Xub and Wei Zhanga* ABSTRACT: A sensitive and simple liquid chromatography–tandem mass spectrometric (HPLC-MS/MS) method for the determination of corilagin in rat plasma has been developed. Samples were prepared with protein precipitation method and analyzed with a triple quadrupole tandem mass spectrometer. We employed negative electrospray ionization as the ionization source and the analytes were detected in multiple reaction monitoring mode. Separation was achieved on a C8 column eluted with mobile phase consisting of methanol–0.1% formic acid in a gradient mode at the flow rate of 0.3 mL/min. The total run time was 7.0 min.This method was proved to have good linearity in the concentration range of 2.5–1000.0 ng/mL. The lower limit of quantification of corilagin was 2.5 ng/mL. The intra- and inter-day relative standard deviationa across three validation runs for four concentration levels were both 5 days. The rat was fasted for 12 h with free access to water before the experiment. All animal experimental procedures in this study were carried out following the guidelines for the care and use of laboratory animals. The rat was orally administered the crude ethanol extract of Phyllanthus urinaria L. (1.0 g/kg) by gavage. Blood samples (approximately 0.3 mL) were collected from the suborbital vein into heparinized centrifuge tubes at 0.0, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0
wileyonlinelibrary.com/journal/bmc
Copyright © 2015 John Wiley & Sons, Ltd.
Biomed. Chromatogr. 2015; 29: 1553–1558
Corilagin Rat Plasma LC-MS/MS and 8.0 h after oral administration. Plasma was separated by centrifugation at 5000 rpm for 10 min and then stored at 80°C until analysis.
in set 2 with that of the standards in set 1. The extraction recoveries were determined by calculating the ratios of the peak areas of the regularly prepared plasma samples in set 3 with that of the spiked post-extraction samples in set 2.
Method validation The current HPLC-MS/MS method was completely validated in biological samples for specificity, linearity, lower limit of quantification (LLOQ), accuracy, precision, extraction recovery, matrix effect (ME) and stability according to the US Food and Drug Administration guidance for bioanalytical method validation. The specificity was evaluated by comparing the chromatograms of the blank rat plasma from six individual rats with the corresponding plasma spiked with corilagin and IS and the plasma sample obtained 4 h after oral administration of Phyllanthus urinaria L. extraction. Plasma samples were quantified using the ratio of the peak area of analyte to that of IS as the assay parameter. Peak area ratios were plotted against analyte concentrations and standard curves were in the form of ‘y = a + bx’. To evaluate linearity, plasma calibration curves were prepared and detected in duplicate on three separate days. The accuracy and precision were also evaluated by determining QC samples at four concentration levels on three different validation days. The accuracy was calculated by (mean observed concentration theoretical concentration)/(theoretical concentration) × 100% and the precision by relative standard deviation (RSD%). Corilagin stability in rat plasma was assessed by analyzing QC samples at concentrations of 5, 50, 250 and 750 ng/mL, respectively, exposed to different time and temperature conditions. The long-term stability was assessed after storage of the test samples at 80°C for 12 days. The freeze–thaw stability was determined after three freeze–thaw cycles, in which the QC samples were thawed completely at room temperature and then refrozen at 80°C for at last 12 h. Autosampler stability was tested by leaving all processed samples in the autosampler at 4°C for 24 h before injection. Matrix effects and extraction recoveries of corilagin were validated in quintuple by comparing the peak areas of the analytes between four different sets of standards of QC concentrations (5, 50, 250 and 750 ng/mL). The first set (set 1) was prepared in 50% methanol. The second set (set 2) was spiked in drug-free plasma extracts and third set (set 3) was prepared in drug-free plasma carrying extracted following ‘Sample preparation’. Matrix effect was assessed by comparing the peak areas of the analyte
Results and discussion Mass spectrometry Negative ionization mode was initially chosen owing to numerous hydroxyl groups (Fig. 1) in both corilagin and rutin. The Q1 full-scan spectra of corilagin and IS dominated by protonated molecules [M H] was significantly observed. When the CID energy was set at 43 and 37 V, the main fragment ion at m/z 633.0–301.0 from corilagin and m/z 609.0–301.0 from rutin showed the highest MS response (Fig. 2). [email protected] Chromatography To decrease the interference of endogenous substances in plasma, a small amount of formic acid was added into the mobile phase. Theoretically, formic acid could improve the resolution of chromatography since most endogenous interference is of an acidic nature but it will in response suppress the MS signal of target compound. Concentrations of 0.01, 0.05, 0.1 and 0.15% formic acid were tested for separation. Finally, 0.1% formic acid was selected because of its high separation efficiency and good MS response. Under these optimum chromatographic conditions, the symmetry of the peak was relative good and the analyte and internal standard were free of interference from endogenous substances. Preparation of plasma samples Protein precipitation is the most widely used biological sample preparation method. In this experiment, methanol, acetonitrile and trichloroacetic acid were all tested for extraction and methanol was finally adopted since it assured high analysis recoveries and stability for the analyzed samples. Method validation Specificity. The specificity tested the ability of the method to differentiate and quantitate the analyte in the presence of other endogenous constituents in the sample and to detect potential
Biomed. Chromatogr. 2015; 29: 1553–1558
Copyright © 2015 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/bmc
1555
Figure 2. Negative ion electrospray mass scan spectra of corilagin (A) and rutin (B).
Q. Q. Chen et al. interferences. The chromatographs of rat blank plasma, the same plasma spiked at the LLOQ of corigagin and the plasma sample obtained at 4 h from a rat after oral administration of Phyllanthus urinaria L. extract are shown in Fig. 3. There was no significant interference or ion suppression from endogenous substances observed at the retention times of the analytes. Typical retention times for corilagin and IS were about 2.42 and 2.68 min, respectively. Linearity of calibration curves and lower limits of quantification. Standard curves were performed in triplicate for each analyte in plasma. In all cases the regression coefficient was >0.99. Corilagin curves showed good linearity over a range of 2.5–1000 ng/mL with a weighting on 1/x2. Typical standard curves were f = 0.0132Ci 0.0118 for corilagin, where f represents the ratio of analyte peak area to that of IS and Ci is the plasma concentrations of analyte. The LLOQ was defined as the lowest concentration on the calibration curve for which an acceptable accuracy of ±15% and a precision
