- Email: [email protected]
MS: Its pharmacokinetic application
Journal of Chromatography B, 992 (2015) 91–95
Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Short Communication
Simultaneous determination of acetaminophen and dihydrocodeine in human plasma by UPLC–MS/MS: Its pharmacokinetic application Xiangjun Qiu a , Dan Lou b , Ding Su a , Zebin Liu a , Pengtao Gao a , Nan-sheng Zhang c,∗ a b c
Medical College of Henan University of Science and Technology, Luoyang 471003, PR China The Second Affiliated Hospital & Yuying Children’s Hospital of Wenzhou Medical University, Wenzhou 325027, PR China The First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325000, PR China
a r t i c l e
i n f o
Article history: Received 26 January 2015 Accepted 18 April 2015 Available online 27 April 2015 Keywords: Acetaminophen Dihydrocodeine UPLC–MS/MS Human plasma Pharmacokinetic
a b s t r a c t An ultra performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) method was developed and validated to determine acetaminophen (AAP) and dihydrocodeine (DHC) in human plasma simultaneously. Plasma samples were prepared using protein precipitation with acetonitrile, the two analytes and the internal standard midazolam were separated on an Acquity UPLC BEH C18 column and mass spectrometric analysis was performed using a QTrap5500 mass spectrometer coupled with an electro-spray ionization (ESI) source in the positive ion mode. The MRM transitions of m/z 151.2 → 110.0 and m/z 302.3 → 199.2 were used to quantify for AAP and DHC, respectively. The linearity of this method was found to be within the concentration range of 50–10000 ng/mL for AAP, and 1–100 ng/mL for DHC in human plasma, respectively. The lower limit of quantification (LLOQ) was 50 ng/mL and 1 ng/mL for AAP and DHC in human plasma, respectively. The relative standard deviations (RSD) of intra and inter precision were less than 10% for both AAP and DHC. The analysis time of per sample was 1.0 min. The developed and validated method was successfully applied to a pharmacokinetic study of AAP (500 mg) with DHC (20 mg) capsule in Chinese healthy volunteers (N = 20). © 2015 Elsevier B.V. All rights reserved.
1. Introduction Acetaminophen (N-acetyl-p-amino-phenol, AAP), also known as paracetamol, is a widespread antipyretic and analgesic accepted as an effective treatment for the relief of pain and fever in adults and children [1–3]. AAP has a low appearance of adverse effects, except from those related to hepatotoxicity but at very high doses [4–6]. Dihydrocodeine (DHC) is a semi-synthetic opioid licensed in most countries to treat moderate to severe pain [7]. However, DHC is sometimes prescribed as well as an alternative to other medications (e.g. methadone and buprenorphine) in substitution or detoxification treatment of opioid misuse [8]. AAP/DHC combination represents the standard medication in the second step of the analgesic ladder and is the most commonly used opioid analgesic for a variety of pain conditions in China [9–11]. The combination of two different drugs acting peripherally and centrally has been specifically used in order to potentiate both efficacy and tolerability [12]. Several chromatographic methods have been developed for the determination of AAP in human plasma including HPLC [13,14], or
with tandem mass spectrometer detector [15–17]. Also, a number of methods have been reported for the analysis of DHC in human plasma including HPLC [18,19], LC–MS [20] or with tandem LC–MS/MS [21,22]. Even though various methods were reported in the literature for estimation of AAP and DHC individually or in combination with other drugs, no method had been reported for simultaneous estimation of these two drugs in human plasma. With recent advances in analysis techniques, ultra performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) has been used increasingly for biological matrix analyses [23,24]. The present paper reports a sensitive UPLC–MS/MS method for the simultaneous determination of AAP and DHC in human plasma which demonstrated a LLOQ of 50 ng/mL for AAP and 1 ng/mL for DHC. The developed and validated method has been successfully applied to a pharmacokinetic study after oral dosing with a fixeddose combination capsule of AAP/DHC (500 mg/20 mg) to healthy volunteers (N = 20) in the fasted state. 2. Materials and methods 2.1. Chemicals and reagents
∗ Corresponding author. Tel.: +86 577 8800 2658. E-mail address: [email protected] (N.-s. Zhang). http://dx.doi.org/10.1016/j.jchromb.2015.04.031 1570-0232/© 2015 Elsevier B.V. All rights reserved.
AAP (purity 98.0%), DHC (purity 98.0%), and midazolam (internal standard, IS, purity 98.0%) were obtained from Sigma (St.
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Louis, MO, USA). Formic acid was of analytical grade and purchased from the Beijing Chemical Reagents Company (Beijing, China). HPLC-grade acetonitrile was purchased from Merck Company (Darmstadt, Germany). Ultra-pure water was prepared by a Millipore Milli-Q purification system (Bedford, MA, USA).
2.2. UPLC–MS/MS conditions Liquid chromatography was performed on an Acquity ultra performance liquid chromatography (UPLC) unit (Waters Corp., Milford, MA) with an Acquity BEH C18 column (2.1 mm × 50 mm, 1.7 m particle size) and inline 0.2 m stainless steel frit filter (Waters Corp., Milford, USA). A gradient program was employed for chromatographic separation with solvent A (0.1% formic acid in water) and solvent B (acetonitrile) as follows: 50–95% B (0–0.3 min), 95–95% B (0.3–0.9 min), 95–50% B (0.9–1.0 min). A subsequent re-equilibration time (1 min) should be performed before next injection. The flow rate was 0.40 mL/min and the injection volume was 6 L. The column and sample temperature were maintained at 40 ◦ C and 4 ◦ C, respectively. An AB Sciex QTRAP 5500 triple quadruple mass spectrometer equipped with an electro-spray ionization (ESI) source (Toronto, Canada) was used for mass spectrometric detection. The quantitative analysis of AAP and DHC in human plasma was performed using multiple reaction monitoring (MRM) method. The dwell time was set to 300 ms for each MRM transition. The MRM transitions were m/z 151.2 → 110.0, m/z 302.3 → 199.2, and m/z 326.2 → 291.1 for AAP, DHC and IS, respectively. The optimal MS parameters were as follows: capillary voltage 4.0 kV, desolvation line (DL) temperature 250 ◦ C, heat block temperature 500 ◦ C, nebulizing gas flow and drying gas flow were 3.0 L/min and 15.0 L/min, respectively. Data acquiring and processing were performed using analyst software (version 1.5, AB Sciex).
2.3. Standard solutions, calibration standards and quality control (QC) sample The stock solutions for AAP (1.00 mg/mL), DHC (1.00 mg/mL) and IS (1.00 mg/mL) were prepared in methanol. Working solutions for calibration and controls were prepared from the stock solutions by dilution with methanol. The IS working solution (300 ng/mL) was prepared from the IS stock solution by dilution using acetonitrile. Calibration standards and QC samples were prepared by spiking appropriate amounts of working solutions into blank human plasma. Final concentrations of the calibration standards were 50, 100, 200, 500, 1000, 2000, 5000, and 10,000 ng/mL for AAP, and 1, 2, 5, 10, 20, 50, 75, and 100 ng/mL for DHC in human plasma. The concentrations of quality-control (QC) samples in plasma were 100, 800, 8000 ng/mL for AAP, and 2, 20, 80 ng/mL for DHC. All stock solutions, working solutions, calibration standards and QCs were immediately stored at −20 ◦ C.
2.4. Sample preparation Before analysis, the plasma sample was thawed to room temperature. In a 1.5 mL centrifuge tube, an aliquot of 200 L of the IS working solution (300 ng/mL in acetonitrile) was added to 100 L of collected plasma sample, and the tubes were vortex mixed for 1.0 min. After centrifugation at 14,900 × g for 10 min, the supernatant (5 L) was injected into the UPLC–MS/MS system for analysis.
2.5. Method validation Before used to determinate the clinical levels, the method was fully validated for specificity, linearity, sensitivity, precision, accuracy, recovery, matrix effect and stability according to the United States Food and Drug Administration (USFDA) guidelines for the validation of a bioanalytical method [25]. A specificity study is designed to investigate whether endogenous substances were observed at the retention time of analytes and IS. Specificity was evaluated by comparing the chromatograms of six different batches of blank human plasma with the corresponding spiked plasma. Calibration curves were constructed according to Section 2.3. The linearity of each calibration curve was determined by plotting the peak area ratio (y) of analytes to IS against the analyte concentration (x) with weighted (1/x2 ) least square linear regression in the concentration range of 50–10000 ng/mL for AAP, and 1–100 ng/mL for DHC. The sensitivity of the method was determined by quantifying the lower limit of quantification (LLOQ). The LLOQ was defined as the lowest acceptable point in the calibration curve which was determined at an acceptable precision (≤20%) and accuracy (≤20%). The recoveries of AAP and DHC were evaluated by comparing peak area ratios of extracted QC samples with those of reference QC solutions reconstituted in blank plasma extracts (n = 6). The recovery of the IS (300 ng/mL) was determined in a similar way. The matrix effect was evaluated by comparing the peak areas of the post-extracted spiked QC samples with those of corresponding standard solutions. The matrix effect of the analytes was determined by analyzing six plasma samples at three concentration levels. The matrix effect of IS was determined using the same procedure at a single concentration of 300 ng/mL. For the evaluation of intra-day precision and accuracy, six replicates QC samples were analyzed at three concentration levels on the same day. For the evaluation of inter-day precision and accuracy, three replicates of QC samples were analyzed at three concentration levels on six consecutive days. Precision was expressed as the percentage relative standard deviations (RSD, %) and accuracy was expressed as was expressed as the relative error (RE, %). The stability experiments were performed to evaluate the stability of the analytes in human plasma under the following conditions: short-term stability at room temperature for 2 h; long-term stability at −20 ◦ C for 28 days; three freeze (−20 ◦ C) – thaw (room temperature) cycles on consecutive days. The extracted QC samples kept in the autosampler at 4 ◦ C for 12 h were analyzed to evaluate post-preparation stability. All stability testing in plasma was determined by analyzing five replicates of QC samples at three concentration levels. The determined concentrations were compared with the nominal values. 2.6. Application The validated bioanalytical method was successfully applied to evaluate the pharmacokinetic study of AAP and DHC in healthy volunteers. The clinical protocol was approved by Medical Ethics Committee of the First Affiliated Hospital of Henan University of Science and Technology prior to the study. Twenty volunteers were given written informed consent to participate in the study according to the principles of the Declaration of Helsinki. The volunteers who submitted the agreements to attend this project were non-smokers and medically examined for the study. The subjects were required to abstain from taking any other drug for 7 days prior to the start of test. They were also demanded not to drink alcohol for 24 h before the beginning of the study until its end. All volunteers were received an oral fixed-dose combination capsule of AAP/DHC (500 mg/20 mg) with 200 mL water. Blood samples
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Table 1 Recovery and matrix effect of AAP, DHC and internal standards (n = 6). Analytes
Concentration added (ng/mL)
Recovery (%)
Matrix effect (%)
Mean ± SD 100 800 8000 2 20 80 300
AAP
DHC IS
80.8 81.5 80.0 84.0 87.8 83.7 89.6
± ± ± ± ± ± ±
5.6 2.7 6.4 3.8 3.8 2.8 4.9
(3 mL) were collected into heparinized tubes at 0.167, 0.333, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 8, 12 and 14 h after oral administration. Blood samples were centrifuged at 4000 × g for 10 min and the plasma was separated and kept frozen at −20 ◦ C until analysis. Plasma concentration vs. time profiles was analyzed using DAS software (version 2.0, Medical University of Wenzhou, China) to estimate the type of compartment model and pharmacokinetic parameters. Data were expressed as mean ± SD. 3. Results and discussion
RSD (%)
Mean ± SD
6.9 3.4 8.0 4.6 4.3 3.4 5.5
97.8 99.8 100.8 99.8 97.8 96.7 103.5
± ± ± ± ± ± ±
RSD (%)
7.0 5.8 5.7 4.1 2.7 6.2 5.4
7.2 5.8 5.6 4.1 2.8 6.4 5.2
improve the sensitivity. However, protein precipitation has the advantages of saving time and simplicity. In this work, protein precipitation was employed owing to the effective and simple process. 3.2. Specificity Fig. 1 shows a typical chromatogram for the blank plasma (Fig. 1A), blank plasma spiked with analytes and IS (Fig. 1B), and human plasma obtained 1.5 h after oral administration of AAP and DHC (Fig. 1C). As shown in Fig. 1, there were no obvious endogenous interferences under the described chromatographic conditions.
3.1. Method development and optimization 3.3. Linearity and sensitivity It was important to optimize chromatographic conditions, mass spectrometry parameters and extraction technique to develop and validate a selective and rapid assay method for simultaneous quantitation of AAP and DHC in human plasma. MS parameters were optimized by infusing standard analyte solution of 1000 ng/mL into the mass spectrometer having electrospray as the ionization source and operating in the multiple reaction monitoring (MRM) mode. The signal intensities obtained in positive mode were much higher than those in negative ion mode since the analytes and internal standard have the ability to accept protons. AAP, DHC and IS gave predominant protonated (M + H)+ parent ions at m/z 151.2, 302.3, and 326.2 ions, respectively in Q1 MS full scan spectra. Fragmentation was initiated using sufficient nitrogen for collision-activated dissociation and by applying 37 V collision energy to break the parent ions. The most abundant ions found in the product ion mass spectrum were m/z 110.0, 199.2, and 291.1 for AAP, DHC and IS, respectively. Declustering potential and collision energy were determined by observing maximum response of the product ion. Electrospray ionization (ESI) was selected as the ionization source as it gave high spectral response for both the analytes and the regression curves obtained were linear. Also, ESI source provided reliable data on method validation and for quantitation of samples from human volunteers. Sample preparation is a key step in the method development. Simultaneous recovery of both the analytes from plasma was difficult. Two kinds of sample treatment procedures were evaluated, including liquid–liquid extraction and protein precipitation technique. Liquid–liquid extraction could provide less interference and
The calibration curves were created by plotting the peak area ratios of the various analytes to internal standard versus nominal concentration of the analytes standards. Both calibration curves were regressed using a linear equation with a weighting factor of 1/x2 . The coefficient of correlation of all the calibration curves was more than 0.99. The typical regression equation of these curves was calculated as follows: AAP, y = (102.1x − 2.2, r = 0.9996); DHC, y = (0.0159x − 0.00684, r = 0.9998). As shown, all the standard calibration curves showed good linearity within the range using least squares regression analysis. The LLOQ values were 50 ng/mL for AAP and 1 ng/mL for DHC. 3.4. Recovery and matrix effect The recoveries and matrix effect of AAP and DHC ranged from 80.8 to 87.8% and from 96.7 to 100.8%, respectively. The recovery and matrix effect of IS were 89.6% and 103.5%, respectively. The detailed results were presented in Table 1. The matrix effect on the ionization of the analytes and IS was not obvious under these conditions. These data indicated that the sample preparation method was satisfactory and resulted in little appreciable matrix effect for the analytes and IS. 3.5. Precision and accuracy The intra- and inter-day precisions and accuracies of low, medium and high QC levels of the analytes were summarized in
Table 2 Precision and accuracy of method for the determination of AAP and DHC in human plasma (n = 6). Analytes
Concentration added (ng/mL)
AAP
100 800 8000
DHC
2 20 80
Intra-day precision (n = 6)
Inter-day precision (n = 6)
Mean ± SD
RSD (%)
104.2 ± 9.3 809.0 ± 24.1 8256.7 ± 242.6
8.9 3.0 2.9
4.2 1.1 3.2
1.9 ± 0.1 19.4 ± 1.4 81.2 ± 4.6
6.1 7.0 5.7
−4.6 −2.9 1.5
RE (%)
Mean ± SD
RSD (%)
RE (%)
97.7 ± 9.5 799.2 ± 21.8 7962.8 ± 317.1
9.7 2.7 4.0
−2.3 −0.1 −0.5
2.1 ± 0.1 20.5 ± 1.6 77.4 ± 2.9
6.6 7.8 3.8
7.0 2.7 −3.3
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Fig. 1. Representative chromatograms of AAP, DHC and IS in human plasma samples. (A) A blank plasma sample; (B) a blank plasma sample spiked with AAP (1.0 g/mL), DHC (50.0 ng/mL) and IS (100.0 ng/mL); (C) a plasma sample from a human 1.5 h after an oral co-administration of AAP (2.2 g/mL) and DHC (144.6 ng/mL).
Fig. 2. Mean plasma concentration–time profiles for AAP (A) and DHC (B) after dosing with 500 mg AAP and 20 mg DHC to healthy volunteers (N = 20) in the fasting state (mean ± SD).
Table 2. The assay values for both intra- and inter-day were found to be within the accepted variable limits. The results showed that the method was accurate and precise for the determination of the two analytes in human plasma. 3.6. Stability The stability of the two analytes in plasma was investigated by analyzing five replicates of QC samples at three concentration levels after short-term storage (room temperature, 2 h), at 4 ◦ C for 12 h after preparation, three freeze-thaw cycles, and long-term storage (−20 ◦ C, 28 days). The RSDs of the mean test responses were within 15% in all stability tests. The results were found to be well within the acceptable limits, suggesting that this analytical method was applicable for routine analysis.
Table 3 Pharmacokinetic parameters of AAP and DHC (500 mg/20 mg capsule) administrated to 20 volunteers. Parameter
AAP
t1/2 (h) Cmax a Tmax (h) AUC0 → 14 b AUC0 → ∞ b
2.79 9.12 0.91 35.91 37.24
a b
DHC ± ± ± ± ±
AAP (g/mL) and DHC (ng/mL). AAP (g/mL h) and DHC (ng/mL h).
0.41 2.50 0.47 7.01 7.56
3.16 61.97 0.81 288.92 307.74
± ± ± ± ±
0.35 9.83 0.17 31.52 33.02
3.7. Application of the method in a pharmacokinetic study A rapid and efficient UPLC–MS/MS method has been widely used in biological and medical research, biological analysis, food safety, environmental monitoring and forensic examination. This experiment is the first to use UPLC–MS/MS method to determine the concentration of AAP and DHC in human plasma and its application to a pharmacokinetic study. The mean pharmacokinetic profiles are illustrated in Fig. 2, and the pharmacokinetic parameters derived from these curves are summarized in Table 3. The plasma concentration of AAP and DHC decreased rapidly and was eliminated from plasma with a terminal half-life of 2.79 ± 0.41 h and 3.16 ± 0.35 h, respectively. The initial rapid declines in the plasma concentration indicate that these compounds might have left the plasma and been distributed into the other tissues, but further studies will be conducted to confirm these findings. 4. Conclusions A UPLC–MS/MS method for the simultaneous determination of AAP and DHC in human plasma was developed and validated. To the best of our knowledge, this is the first report of the simultaneous determination of AAP and DHC levels in human plasma using an UPLC–MS/MS method. The advantages of this assay include simple sample preparation procedures, short analysis time (1.0 min per sample), and high sensitivity which rendered the method fits for the purpose of its application to measure concentration–time profiles for bioavailability, pharmacokinetic and bioequivalence decision
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Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Short Communication
Simultaneous determination of acetaminophen and dihydrocodeine in human plasma by UPLC–MS/MS: Its pharmacokinetic application Xiangjun Qiu a , Dan Lou b , Ding Su a , Zebin Liu a , Pengtao Gao a , Nan-sheng Zhang c,∗ a b c
Medical College of Henan University of Science and Technology, Luoyang 471003, PR China The Second Affiliated Hospital & Yuying Children’s Hospital of Wenzhou Medical University, Wenzhou 325027, PR China The First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325000, PR China
a r t i c l e
i n f o
Article history: Received 26 January 2015 Accepted 18 April 2015 Available online 27 April 2015 Keywords: Acetaminophen Dihydrocodeine UPLC–MS/MS Human plasma Pharmacokinetic
a b s t r a c t An ultra performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) method was developed and validated to determine acetaminophen (AAP) and dihydrocodeine (DHC) in human plasma simultaneously. Plasma samples were prepared using protein precipitation with acetonitrile, the two analytes and the internal standard midazolam were separated on an Acquity UPLC BEH C18 column and mass spectrometric analysis was performed using a QTrap5500 mass spectrometer coupled with an electro-spray ionization (ESI) source in the positive ion mode. The MRM transitions of m/z 151.2 → 110.0 and m/z 302.3 → 199.2 were used to quantify for AAP and DHC, respectively. The linearity of this method was found to be within the concentration range of 50–10000 ng/mL for AAP, and 1–100 ng/mL for DHC in human plasma, respectively. The lower limit of quantification (LLOQ) was 50 ng/mL and 1 ng/mL for AAP and DHC in human plasma, respectively. The relative standard deviations (RSD) of intra and inter precision were less than 10% for both AAP and DHC. The analysis time of per sample was 1.0 min. The developed and validated method was successfully applied to a pharmacokinetic study of AAP (500 mg) with DHC (20 mg) capsule in Chinese healthy volunteers (N = 20). © 2015 Elsevier B.V. All rights reserved.
1. Introduction Acetaminophen (N-acetyl-p-amino-phenol, AAP), also known as paracetamol, is a widespread antipyretic and analgesic accepted as an effective treatment for the relief of pain and fever in adults and children [1–3]. AAP has a low appearance of adverse effects, except from those related to hepatotoxicity but at very high doses [4–6]. Dihydrocodeine (DHC) is a semi-synthetic opioid licensed in most countries to treat moderate to severe pain [7]. However, DHC is sometimes prescribed as well as an alternative to other medications (e.g. methadone and buprenorphine) in substitution or detoxification treatment of opioid misuse [8]. AAP/DHC combination represents the standard medication in the second step of the analgesic ladder and is the most commonly used opioid analgesic for a variety of pain conditions in China [9–11]. The combination of two different drugs acting peripherally and centrally has been specifically used in order to potentiate both efficacy and tolerability [12]. Several chromatographic methods have been developed for the determination of AAP in human plasma including HPLC [13,14], or
with tandem mass spectrometer detector [15–17]. Also, a number of methods have been reported for the analysis of DHC in human plasma including HPLC [18,19], LC–MS [20] or with tandem LC–MS/MS [21,22]. Even though various methods were reported in the literature for estimation of AAP and DHC individually or in combination with other drugs, no method had been reported for simultaneous estimation of these two drugs in human plasma. With recent advances in analysis techniques, ultra performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) has been used increasingly for biological matrix analyses [23,24]. The present paper reports a sensitive UPLC–MS/MS method for the simultaneous determination of AAP and DHC in human plasma which demonstrated a LLOQ of 50 ng/mL for AAP and 1 ng/mL for DHC. The developed and validated method has been successfully applied to a pharmacokinetic study after oral dosing with a fixeddose combination capsule of AAP/DHC (500 mg/20 mg) to healthy volunteers (N = 20) in the fasted state. 2. Materials and methods 2.1. Chemicals and reagents
∗ Corresponding author. Tel.: +86 577 8800 2658. E-mail address: [email protected] (N.-s. Zhang). http://dx.doi.org/10.1016/j.jchromb.2015.04.031 1570-0232/© 2015 Elsevier B.V. All rights reserved.
AAP (purity 98.0%), DHC (purity 98.0%), and midazolam (internal standard, IS, purity 98.0%) were obtained from Sigma (St.
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Louis, MO, USA). Formic acid was of analytical grade and purchased from the Beijing Chemical Reagents Company (Beijing, China). HPLC-grade acetonitrile was purchased from Merck Company (Darmstadt, Germany). Ultra-pure water was prepared by a Millipore Milli-Q purification system (Bedford, MA, USA).
2.2. UPLC–MS/MS conditions Liquid chromatography was performed on an Acquity ultra performance liquid chromatography (UPLC) unit (Waters Corp., Milford, MA) with an Acquity BEH C18 column (2.1 mm × 50 mm, 1.7 m particle size) and inline 0.2 m stainless steel frit filter (Waters Corp., Milford, USA). A gradient program was employed for chromatographic separation with solvent A (0.1% formic acid in water) and solvent B (acetonitrile) as follows: 50–95% B (0–0.3 min), 95–95% B (0.3–0.9 min), 95–50% B (0.9–1.0 min). A subsequent re-equilibration time (1 min) should be performed before next injection. The flow rate was 0.40 mL/min and the injection volume was 6 L. The column and sample temperature were maintained at 40 ◦ C and 4 ◦ C, respectively. An AB Sciex QTRAP 5500 triple quadruple mass spectrometer equipped with an electro-spray ionization (ESI) source (Toronto, Canada) was used for mass spectrometric detection. The quantitative analysis of AAP and DHC in human plasma was performed using multiple reaction monitoring (MRM) method. The dwell time was set to 300 ms for each MRM transition. The MRM transitions were m/z 151.2 → 110.0, m/z 302.3 → 199.2, and m/z 326.2 → 291.1 for AAP, DHC and IS, respectively. The optimal MS parameters were as follows: capillary voltage 4.0 kV, desolvation line (DL) temperature 250 ◦ C, heat block temperature 500 ◦ C, nebulizing gas flow and drying gas flow were 3.0 L/min and 15.0 L/min, respectively. Data acquiring and processing were performed using analyst software (version 1.5, AB Sciex).
2.3. Standard solutions, calibration standards and quality control (QC) sample The stock solutions for AAP (1.00 mg/mL), DHC (1.00 mg/mL) and IS (1.00 mg/mL) were prepared in methanol. Working solutions for calibration and controls were prepared from the stock solutions by dilution with methanol. The IS working solution (300 ng/mL) was prepared from the IS stock solution by dilution using acetonitrile. Calibration standards and QC samples were prepared by spiking appropriate amounts of working solutions into blank human plasma. Final concentrations of the calibration standards were 50, 100, 200, 500, 1000, 2000, 5000, and 10,000 ng/mL for AAP, and 1, 2, 5, 10, 20, 50, 75, and 100 ng/mL for DHC in human plasma. The concentrations of quality-control (QC) samples in plasma were 100, 800, 8000 ng/mL for AAP, and 2, 20, 80 ng/mL for DHC. All stock solutions, working solutions, calibration standards and QCs were immediately stored at −20 ◦ C.
2.4. Sample preparation Before analysis, the plasma sample was thawed to room temperature. In a 1.5 mL centrifuge tube, an aliquot of 200 L of the IS working solution (300 ng/mL in acetonitrile) was added to 100 L of collected plasma sample, and the tubes were vortex mixed for 1.0 min. After centrifugation at 14,900 × g for 10 min, the supernatant (5 L) was injected into the UPLC–MS/MS system for analysis.
2.5. Method validation Before used to determinate the clinical levels, the method was fully validated for specificity, linearity, sensitivity, precision, accuracy, recovery, matrix effect and stability according to the United States Food and Drug Administration (USFDA) guidelines for the validation of a bioanalytical method [25]. A specificity study is designed to investigate whether endogenous substances were observed at the retention time of analytes and IS. Specificity was evaluated by comparing the chromatograms of six different batches of blank human plasma with the corresponding spiked plasma. Calibration curves were constructed according to Section 2.3. The linearity of each calibration curve was determined by plotting the peak area ratio (y) of analytes to IS against the analyte concentration (x) with weighted (1/x2 ) least square linear regression in the concentration range of 50–10000 ng/mL for AAP, and 1–100 ng/mL for DHC. The sensitivity of the method was determined by quantifying the lower limit of quantification (LLOQ). The LLOQ was defined as the lowest acceptable point in the calibration curve which was determined at an acceptable precision (≤20%) and accuracy (≤20%). The recoveries of AAP and DHC were evaluated by comparing peak area ratios of extracted QC samples with those of reference QC solutions reconstituted in blank plasma extracts (n = 6). The recovery of the IS (300 ng/mL) was determined in a similar way. The matrix effect was evaluated by comparing the peak areas of the post-extracted spiked QC samples with those of corresponding standard solutions. The matrix effect of the analytes was determined by analyzing six plasma samples at three concentration levels. The matrix effect of IS was determined using the same procedure at a single concentration of 300 ng/mL. For the evaluation of intra-day precision and accuracy, six replicates QC samples were analyzed at three concentration levels on the same day. For the evaluation of inter-day precision and accuracy, three replicates of QC samples were analyzed at three concentration levels on six consecutive days. Precision was expressed as the percentage relative standard deviations (RSD, %) and accuracy was expressed as was expressed as the relative error (RE, %). The stability experiments were performed to evaluate the stability of the analytes in human plasma under the following conditions: short-term stability at room temperature for 2 h; long-term stability at −20 ◦ C for 28 days; three freeze (−20 ◦ C) – thaw (room temperature) cycles on consecutive days. The extracted QC samples kept in the autosampler at 4 ◦ C for 12 h were analyzed to evaluate post-preparation stability. All stability testing in plasma was determined by analyzing five replicates of QC samples at three concentration levels. The determined concentrations were compared with the nominal values. 2.6. Application The validated bioanalytical method was successfully applied to evaluate the pharmacokinetic study of AAP and DHC in healthy volunteers. The clinical protocol was approved by Medical Ethics Committee of the First Affiliated Hospital of Henan University of Science and Technology prior to the study. Twenty volunteers were given written informed consent to participate in the study according to the principles of the Declaration of Helsinki. The volunteers who submitted the agreements to attend this project were non-smokers and medically examined for the study. The subjects were required to abstain from taking any other drug for 7 days prior to the start of test. They were also demanded not to drink alcohol for 24 h before the beginning of the study until its end. All volunteers were received an oral fixed-dose combination capsule of AAP/DHC (500 mg/20 mg) with 200 mL water. Blood samples
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Table 1 Recovery and matrix effect of AAP, DHC and internal standards (n = 6). Analytes
Concentration added (ng/mL)
Recovery (%)
Matrix effect (%)
Mean ± SD 100 800 8000 2 20 80 300
AAP
DHC IS
80.8 81.5 80.0 84.0 87.8 83.7 89.6
± ± ± ± ± ± ±
5.6 2.7 6.4 3.8 3.8 2.8 4.9
(3 mL) were collected into heparinized tubes at 0.167, 0.333, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 8, 12 and 14 h after oral administration. Blood samples were centrifuged at 4000 × g for 10 min and the plasma was separated and kept frozen at −20 ◦ C until analysis. Plasma concentration vs. time profiles was analyzed using DAS software (version 2.0, Medical University of Wenzhou, China) to estimate the type of compartment model and pharmacokinetic parameters. Data were expressed as mean ± SD. 3. Results and discussion
RSD (%)
Mean ± SD
6.9 3.4 8.0 4.6 4.3 3.4 5.5
97.8 99.8 100.8 99.8 97.8 96.7 103.5
± ± ± ± ± ± ±
RSD (%)
7.0 5.8 5.7 4.1 2.7 6.2 5.4
7.2 5.8 5.6 4.1 2.8 6.4 5.2
improve the sensitivity. However, protein precipitation has the advantages of saving time and simplicity. In this work, protein precipitation was employed owing to the effective and simple process. 3.2. Specificity Fig. 1 shows a typical chromatogram for the blank plasma (Fig. 1A), blank plasma spiked with analytes and IS (Fig. 1B), and human plasma obtained 1.5 h after oral administration of AAP and DHC (Fig. 1C). As shown in Fig. 1, there were no obvious endogenous interferences under the described chromatographic conditions.
3.1. Method development and optimization 3.3. Linearity and sensitivity It was important to optimize chromatographic conditions, mass spectrometry parameters and extraction technique to develop and validate a selective and rapid assay method for simultaneous quantitation of AAP and DHC in human plasma. MS parameters were optimized by infusing standard analyte solution of 1000 ng/mL into the mass spectrometer having electrospray as the ionization source and operating in the multiple reaction monitoring (MRM) mode. The signal intensities obtained in positive mode were much higher than those in negative ion mode since the analytes and internal standard have the ability to accept protons. AAP, DHC and IS gave predominant protonated (M + H)+ parent ions at m/z 151.2, 302.3, and 326.2 ions, respectively in Q1 MS full scan spectra. Fragmentation was initiated using sufficient nitrogen for collision-activated dissociation and by applying 37 V collision energy to break the parent ions. The most abundant ions found in the product ion mass spectrum were m/z 110.0, 199.2, and 291.1 for AAP, DHC and IS, respectively. Declustering potential and collision energy were determined by observing maximum response of the product ion. Electrospray ionization (ESI) was selected as the ionization source as it gave high spectral response for both the analytes and the regression curves obtained were linear. Also, ESI source provided reliable data on method validation and for quantitation of samples from human volunteers. Sample preparation is a key step in the method development. Simultaneous recovery of both the analytes from plasma was difficult. Two kinds of sample treatment procedures were evaluated, including liquid–liquid extraction and protein precipitation technique. Liquid–liquid extraction could provide less interference and
The calibration curves were created by plotting the peak area ratios of the various analytes to internal standard versus nominal concentration of the analytes standards. Both calibration curves were regressed using a linear equation with a weighting factor of 1/x2 . The coefficient of correlation of all the calibration curves was more than 0.99. The typical regression equation of these curves was calculated as follows: AAP, y = (102.1x − 2.2, r = 0.9996); DHC, y = (0.0159x − 0.00684, r = 0.9998). As shown, all the standard calibration curves showed good linearity within the range using least squares regression analysis. The LLOQ values were 50 ng/mL for AAP and 1 ng/mL for DHC. 3.4. Recovery and matrix effect The recoveries and matrix effect of AAP and DHC ranged from 80.8 to 87.8% and from 96.7 to 100.8%, respectively. The recovery and matrix effect of IS were 89.6% and 103.5%, respectively. The detailed results were presented in Table 1. The matrix effect on the ionization of the analytes and IS was not obvious under these conditions. These data indicated that the sample preparation method was satisfactory and resulted in little appreciable matrix effect for the analytes and IS. 3.5. Precision and accuracy The intra- and inter-day precisions and accuracies of low, medium and high QC levels of the analytes were summarized in
Table 2 Precision and accuracy of method for the determination of AAP and DHC in human plasma (n = 6). Analytes
Concentration added (ng/mL)
AAP
100 800 8000
DHC
2 20 80
Intra-day precision (n = 6)
Inter-day precision (n = 6)
Mean ± SD
RSD (%)
104.2 ± 9.3 809.0 ± 24.1 8256.7 ± 242.6
8.9 3.0 2.9
4.2 1.1 3.2
1.9 ± 0.1 19.4 ± 1.4 81.2 ± 4.6
6.1 7.0 5.7
−4.6 −2.9 1.5
RE (%)
Mean ± SD
RSD (%)
RE (%)
97.7 ± 9.5 799.2 ± 21.8 7962.8 ± 317.1
9.7 2.7 4.0
−2.3 −0.1 −0.5
2.1 ± 0.1 20.5 ± 1.6 77.4 ± 2.9
6.6 7.8 3.8
7.0 2.7 −3.3
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Fig. 1. Representative chromatograms of AAP, DHC and IS in human plasma samples. (A) A blank plasma sample; (B) a blank plasma sample spiked with AAP (1.0 g/mL), DHC (50.0 ng/mL) and IS (100.0 ng/mL); (C) a plasma sample from a human 1.5 h after an oral co-administration of AAP (2.2 g/mL) and DHC (144.6 ng/mL).
Fig. 2. Mean plasma concentration–time profiles for AAP (A) and DHC (B) after dosing with 500 mg AAP and 20 mg DHC to healthy volunteers (N = 20) in the fasting state (mean ± SD).
Table 2. The assay values for both intra- and inter-day were found to be within the accepted variable limits. The results showed that the method was accurate and precise for the determination of the two analytes in human plasma. 3.6. Stability The stability of the two analytes in plasma was investigated by analyzing five replicates of QC samples at three concentration levels after short-term storage (room temperature, 2 h), at 4 ◦ C for 12 h after preparation, three freeze-thaw cycles, and long-term storage (−20 ◦ C, 28 days). The RSDs of the mean test responses were within 15% in all stability tests. The results were found to be well within the acceptable limits, suggesting that this analytical method was applicable for routine analysis.
Table 3 Pharmacokinetic parameters of AAP and DHC (500 mg/20 mg capsule) administrated to 20 volunteers. Parameter
AAP
t1/2 (h) Cmax a Tmax (h) AUC0 → 14 b AUC0 → ∞ b
2.79 9.12 0.91 35.91 37.24
a b
DHC ± ± ± ± ±
AAP (g/mL) and DHC (ng/mL). AAP (g/mL h) and DHC (ng/mL h).
0.41 2.50 0.47 7.01 7.56
3.16 61.97 0.81 288.92 307.74
± ± ± ± ±
0.35 9.83 0.17 31.52 33.02
3.7. Application of the method in a pharmacokinetic study A rapid and efficient UPLC–MS/MS method has been widely used in biological and medical research, biological analysis, food safety, environmental monitoring and forensic examination. This experiment is the first to use UPLC–MS/MS method to determine the concentration of AAP and DHC in human plasma and its application to a pharmacokinetic study. The mean pharmacokinetic profiles are illustrated in Fig. 2, and the pharmacokinetic parameters derived from these curves are summarized in Table 3. The plasma concentration of AAP and DHC decreased rapidly and was eliminated from plasma with a terminal half-life of 2.79 ± 0.41 h and 3.16 ± 0.35 h, respectively. The initial rapid declines in the plasma concentration indicate that these compounds might have left the plasma and been distributed into the other tissues, but further studies will be conducted to confirm these findings. 4. Conclusions A UPLC–MS/MS method for the simultaneous determination of AAP and DHC in human plasma was developed and validated. To the best of our knowledge, this is the first report of the simultaneous determination of AAP and DHC levels in human plasma using an UPLC–MS/MS method. The advantages of this assay include simple sample preparation procedures, short analysis time (1.0 min per sample), and high sensitivity which rendered the method fits for the purpose of its application to measure concentration–time profiles for bioavailability, pharmacokinetic and bioequivalence decision
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