Journal of Chromatographic Science, 2016, Vol. 54, No. 4, 569–573 doi: 10.1093/chromsci/bmv188 Advance Access Publication Date: 20 December 2015 Article
Article
UPLC–MS-MS Determination of Dihydrocodeine in Human Plasma and Its Application to a Pharmacokinetic Study Wei-min Zhang1,*, Yan-pei Duan2, Wei Li1, Jian-fei Qiu3, and Zhi-yin Zhang3 1
The First Affiliated Hospital of Henan University of Science and Technology, Luoyang, Henan 471003, PR China, Department of Pharmacy, Xinxiang Central Hospital, Xinxiang, Henan 453000, PR China, and 3Medical College of Henan University of Science and Technology, Luoyang, Henan 471003, PR China
2
*Author to whom correspondence should be addressed. Email: [email protected] Received 20 December 2014; Revised 23 October 2015
Abstract A sensitive and rapid ultra performance liquid chromatography tandem mass spectrometry (UPLC– MS-MS) method was developed to determine dihydrocodeine (DHC) in human plasma using diazepam as the internal standard (IS). Sample preparation was accomplished through a liquid–liquid extraction procedure with ethyl acetate. The analyte and IS were separated on an Acquity UPLC BEH C18 column (2.1 × 50 mm, 1.7 μm) with the mobile phase of acetonitrile and 1% formic acid in water with gradient elution at a flow rate of 0.4 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer equipped with positive-ion electrospray ionization by multiple reaction monitoring (MRM) of the transitions at m/z 302.3 → 199.2 for DHC and m/z 285.1 → 193.1 for IS. The linearity of this method was found to be within the concentration range of 0.5–100 ng/mL with a lower limit of quantification of 0.5 ng/mL. The overall run time was 4.0 min. The method herein described was superior to previous methods and was successfully applied to the pharmacokinetic study of DHC in healthy Chinese volunteers after oral administration.
Introduction Dihydrocodeine (DHC, Figure 1) is a semi-synthetic opioid, licensed in most countries to treat moderate to severe pain (1, 2). It is a so-called “weak” opioid that can be used if nonopioid analgesics such as paracetamol and nonsteroidal anti-inflammatory drugs provide insufficient pain control (2, 3). Bioavailability of DHC after oral administration is ∼20%, which indicates that its analgesic efficacy after oral administration is slightly stronger than that of codeine (bioavailability after oral administration approximately equals 30–40%) (4). After DHC oral administration, the maximal serum concentration appears after 1.7 h, plasma half-life is 3.5–5.5 h and analgesia period is 4 h. However, there is little data available on DHC pharmacokinetics. Several methods have been published for the determination of DHC in biological matrices, including high-performance liquid chromatography (HPLC) (5–9), high-performance liquid chromatography tandem mass spectrometry (HPLC–MS-MS) (10–12) and gas chromatography–mass spectrometry (GC–MS) (13–16). However, these
methods have their own disadvantages, such as the lack of sensitivity, complex analyzing processes, long time requirement for preparation of samples and long chromatographic run times, which are not preferable when dealing with a large number of samples. Moreover, no publication has described the quantitative analysis of DHC in the biological fluid using ultra performance liquid chromatography tandem mass spectrometry (UPLC–MS-MS). Therefore, a novel, simple and rapid method for the measurement of DHC concentration in human plasma sample is still required for therapeutic drug monitoring. UPLC–MS-MS has been considered as a faster and more efficient analytical tool compared with current chromatography (17–19). In this study, a UPLC–MS-MS method for the determination of DHC using diazepam as an internal standard (IS) was developed. This new method has been fully validated in terms of selectivity, linearity, lower limit of quantification (LLOQ), accuracy, precision, stability, matrix effect and recovery. It has been successfully applied in a pharmacokinetic study conducted in Chinese healthy male volunteers.
© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
569
570
Zhang et al. for dilution. All stock solutions, working solutions, calibration standards and QCs were immediately stored at −20°C.
Sample preparation
Figure 1. The chemical structures of DHC and IS in this study: (A) DHC and (B) diazepam (IS).
Experimental Chemical materials DHC and diazepam (IS) were obtained from Sigma (St. Louis, MO, USA). Acetonitrile and methanol were of HPLC grade and purchased from Merck Company (Darmstadt, Germany). HPLC grade water was obtained using a Milli Q system (Millipore, Bedford, USA).
UPLC–MS-MS conditions Liquid chromatography was performed on an Acquity ultra performance liquid chromatography (UPLC) unit (Waters Corp., Milford, MA, USA) with an Acquity BEH C18 column (2.1 × 50 mm, 1.7 μm particle size) and inline 0.2 μm stainless steel frit filter (Waters Corp.). A gradient elution program was conducted for chromatographic separation with mobile phase A (acetonitrile), and mobile phase B (0.1% formic acid in water) as follows: 0–0.5 min (20–75% A), 0.5–2.0 min (75–75% A), 2.0–2.1 min (75–20% A) and 2.1– 4.0 min (20–20% A). The flow rate was 0.4 mL/min. The overall run time was 4.0 min. A XEVO TQD triple quadrupole mass spectrometer equipped with an electro-spray ionization (ESI) source was used for mass spectrometric detection. The detection was operated in the multiple reaction monitoring (MRM) mode under unit mass resolution in the mass analyzers. The MRM transitions were m/z 302.3 → 199.2 and m/z 285.1 → 193.1 for DHC and IS, respectively. After optimization, the source parameters were set as follows: curtain gas, 35 psig; nebulizer gas, 50 psig; turbo gas, 60 psig; ion spray voltage, 4.0 kV and temperature, 500°C. Data acquisition and processing were performed using Analyst Software (Waters Corp.).
Standard solutions, calibration standards and quality control sample The stock solution of DHC that was used to make the calibration standards and quality control (QC) samples was prepared by dissolving 10 mg in 10 mL methanol to obtain a concentration of 1.0 mg/mL. The stock solution was further diluted with methanol to obtain working solutions at several concentration levels. Calibration standards and QC samples in plasma were prepared by diluting the corresponding working solutions with blank human plasma. Final concentrations of the calibration standards were 0.5, 1, 2, 5, 10, 20, 50 and 100 ng/mL for DHC in human plasma. The concentrations of QC samples in plasma were 1, 8 and 80 ng/mL for DHC. IS stock solution was made at an initial concentration of 1 mg/mL. The IS working solution (200 ng/mL) was made from the stock solution using methanol
Before analysis, the plasma samples were thawed to ambient temperature. In a 1.5-mL centrifuge tube, an aliquot of 20 µL of the IS working solution (200 ng/mL) and 100 µL of NaOH (1 mol/L) were added to 200 µL of the collected plasma sample followed by the addition of 1.0 mL ethyl acetate. The tubes were vortex mixed for 1.0 min. After centrifugation at 8,000 × g for 10 min, the supernatant organic layer was transferred into a 1.5-mL centrifuge tube and dried under nitrogen stream at 40°C. The dried residue was reconstituted in 80 µL of acetonitrile and water (1 : 1, v/v), and a 3-µL aliquot of this was injected into UPLC–MS-MS system for the analysis.
Method validation Before using this method to determinate DHC in human plasma, the method was fully validated for specificity, linearity, precision, accuracy, recovery, matrix effect and stability according the United States Food and Drug Administration bioanalytical method validation guidances (20). Specificity was determined by analysis of blank human plasma samples from six different volunteers, every blank sample was handled by the procedure described in the “Sample preparation” section and confirmed that endogenous substances did not have the possible interference with the analyte and the IS. To evaluate the linearity, calibration standards of nine concentrations of DHC (0.5–100 ng/mL) were separately extracted and assayed on three separate days. The linearity for DHC was investigated by weighted (1/x 2) least-squares linear regression of peak area ratios against concentrations. The sensitivity of the method was determined by quantifying the LLOQ. The LLOQ was defined as the lowest acceptable point in the calibration curve, which was determined at an acceptable precision and accuracy. To determine the matrix effect, six different blank human samples were utilized to prepare QC samples and used for assessing the lot-to-lot matrix effect. Matrix effect was evaluated at three QC levels by comparing the peak areas of analytes obtained from plasma samples spiked with analytes after extraction to those of the pure standard solutions at the same concentrations. The matrix effect of IS was evaluated at the working concentration (50 ng/mL) in the same manner. The precision and accuracy of the method were assessed by determination of QC samples in plasma at different concentrations (1, 8 and 80 ng/mL) on three separate days. Precision was expressed as % relative standard deviation (RSD) and accuracy was expressed as % relative error (RE) between the measured and nominal value. The precision for QC samples was within 15%, and accuracy was between −15 and 15%. Extraction recovery experiments, which showed an ability to extract the analyte from the test biological samples, were evaluated by comparing the peak areas obtained from extracted QC samples with nonprocessed standard solutions at three concentrations at the same concentration. Recovery of IS was determined at the working concentration (50 ng/mL) similarly. The stability in human plasma was tested by analyzing five replicates of plasma samples at three concentration levels (1, 8 and 80 ng/mL) under different conditions. The short-term stability was determined after the exposure of the spiked samples at ambient temperature for 3 h, and the ready-to-inject samples (after extraction) in the autosampler at 4°C for 24 h. The freeze–thaw stability was evaluated
571
UPLC–MS-MS Determination of DHC in Human Plasma after three complete freeze–thaw cycles (−20 to 25°C) on consecutive days. The long-term stability was assessed after storage of the standard spiked plasma samples at −20°C for 28 days. Samples were considered to be stable if assay values were within the acceptable limits of accuracy (RE % ≤ ±15%) and precision (RSD % ≤ 15%).
Application to a pharmacokinetic study The present method was applied to a pharmacokinetic study after an oral administration of 20 mg DHC tablets to Chinese male volunteers. The clinical protocol was approved by Medical Ethics Committee of Henan University of Science and Technology prior to the study. Twenty volunteers (aged 21–28 years, weighing 50–70 kg, and height 156– 180 cm) signed the informed consent form and participated in the study according to the principles of the Declaration of Helsinki. The volunteers who submitted the agreements to attend this project were medically examined for the pharmacokinetic study of DHC. The subjects were required to abstain from taking any other drug for 7 days prior to the start of test. They were also demanded to abstain from smoking or drinking alcohol for 24 h before the beginning of the study until its end. All volunteers received an oral dose of 20 mg DHC with 200 mL water. Blood samples (3 mL) were collected into heparinized tubes before and 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 4,000 × g for 8 min, and the plasma was separated and kept frozen at −20°C until analysis.
Data analysis Plasma concentration versus time profiles were analyzed using DAS (Drug and Statistics Software; version 2.0, Henan University of Science and Technology, China) to estimate the type of compartment model and pharmacokinetic parameters. Data were expressed as mean ± SD.
Results Specificity and matrix effect UPLC–MS-MS chromatogram of the analytes in human plasma samples is shown in Figure 2. The retention times of DHC and IS are 1.17 and 1.38 min, respectively. Compared with chromatogram of blank blood sample, no interference of endogenous peaks was observed. The matrix effects for DHC at concentrations of 1, 8 and 80 ng/mL were measured to be 108.4, 110.5 and 93.6% (n = 6), respectively. The ME for IS (50 ng/mL) was 97.3% (n = 6). No apparent matrix
effect was found to affect the determination of DHC and IS in plasma. As a result, the matrix effect from plasma was negligible in this method.
Linearity and sensitivity The peak area ratios of DHC/IS versus the nominal concentrations of DHC showed a good linear relationship over the concentration ranges of 0.5–100 ng/mL in human plasma. A typical regression equation for the calibration curve resulted in an equation of the line of: y = 0.0174x – 0.00792, where y represents the peak area ratios of DHC to the IS and x represents plasma concentrations of the analyte. The LLOQ in human plasma was 0.5 ng/mL with the RSD and RE of 9.5 and 7.3%, respectively.
Precision, accuracy and recovery The intra- and interday precision and accuracy of the method were determined from the analysis of QC samples at three different concentrations (1, 8 and 80 ng/mL) for each biological matrix. The method was reliable and reproducible because RSD % was below 10% and RE % was between −10.5 and 9.7% for all the investigated concentrations of DHC in human plasma. The recovery was calculated by comparing the mean peak areas of the analyte spiked before extraction divided by the areas of analyte samples spiked after extraction and multiplied by 100%. The recovery in plasma ranged from 75.7 to 85.8% for DHC. The recovery of IS (50 ng/mL) in plasma was 83.1%. Assay performance data are presented in Table I. The above results demonstrated that the values were within the acceptable range, and the method was accurate and precise.
Stability Stability tests were performed at the low, medium and high QC samples with five determinations for each under different storage conditions. The RSDs of the mean test responses were within 15% in all stability tests. There was no effect on the quantitation for plasma samples kept at ambient temperature for 3 h and at 4°C for 24 h. No significant degradation was observed when samples of DHC were taken through three freeze (−20°C)–thaw (ambient temperature) cycles. As a result, DHC in samples were stable at −20°C for 28 days.
Application of the method in a pharmacokinetic study The method described above was successfully applied to determine the concentration of DHC in human plasma. After an oral administration of 20 mg DHC tablets, the main pharmacokinetic parameters of DHC
Figure 2. Representative chromatograms of DHC and IS in human plasma samples. (A) A blank plasma sample; (B) a blank plasma sample spiked with DHC and IS and (C) a plasma sample from a person 0.5 h after an oral administration of 20 mg. This figure is available in black and white in print and in color at JCS online.
572
Zhang et al.
Table I. Precision, Accuracy and Recovery for DHC of QC Samples in Human Plasma (n = 6)
Table II. Pharmacokinetic Parameters of DHC After Oral Administration of 20 mg to 20 Chinese Healthy Male Volunteers
Concentration (ng/mL)
RSD%
Parameters
Mean ± SD
Intraday
Interday
Intraday
Interday
0.5 1 8 80
7.5 8.4 4.7 6.3
8.8 9.3 7.8 5.5
9.7 −10.5 7.4 −6.3
−11.3 8.1 −4.8 9.7
t1/2 (h) Cmax (ng/mL) Tmax (h) AUC0→14 (ng/mL h) AUC0→∞ (ng/mL h)
3.146 ± 0.356 60.089 ± 8.318 0.844 ± 0.221 285.394 ± 28.606 301.643 ± 31.051
RE%
Recovery (%) 77.5 80.4 75.7 84.8
mobile phases, acetonitrile–water (containing 0.1% formic acid) was found to be optimal for this study, which provided symmetric peak shapes of the analyte and IS. The total time required for the chromatographic run was 4.0 min. The addition of 0.1% formic acid helped to obtain improved signals.
Conclusion
Figure 3. Plasma concentration versus time curves of DHC after a single oral administration of 20 mg to 20 Chinese healthy male volunteers.
for 20 volunteers were estimated. The mean plasma concentration– time curve of DHC is displayed in Figure 3, and the main pharmacokinetic parameters of DHC were calculated and are summarized in Table II. Compared with recently published articles describing the pharmacokinetic profiles of DHC in healthy volunteers, the pharmacokinetic parameters of DHC obtained in the study were generally similar (7).
Discussion In this study, different sample treatment approaches were investigated to achieve a simple and inexpensive extraction procedure. Protein precipitation extraction was initially tried, but low DHC concentration was not detectable during our method development. Then, a liquid– liquid extraction (LLE) technique was investigated and found to be more sensitive. LLE methods using a mixture of organic solvents have been investigated for the extraction of DHC from biological samples. In this study, different organic solvents including diethyl ether, chloroform and ethyl acetate were investigated. Among them, ethyl acetate was found to be optimal solvent for extraction yielding satisfactory results in terms of both sample cleaning and the analyte extraction recovery. Moreover, the boiling point of ethyl acetate is low, so it was evaporated to dryness more quickly. Hence, ethyl acetate was chosen as the extraction solvent. This one-step extraction with ethyl acetate is cheaper than the reported method, which makes the method suitable for low-cost clinical study (16). Further optimization in chromatography conditions was performed to test different mobile phases. In our assessment of different
An UPLC–MS-MS method for the determination of DHC in human plasma was developed and validated. To the best of our knowledge, this is the first report of the determination of the DHC level in human plasma using an UPLC–MS-MS method. Compared with the analytical methods reported in the literature, the method offered superior sample preparation with LLE with organic solvent to precipitation of plasma protein and a shorter run time of 4.0 min. The method meets the requirement of high sample throughput in bioanalysis and has been successfully applied to the pharmacokinetic study of DHC tablets in healthy volunteers.
References 1. Zamparutti, G., Schifano, F., Corkery, J.M., Oyefeso, A., Ghodse, A.H.; Deaths of opiate/opioid misusers involving dihydrocodeine, UK, 1997– 2007; British Journal of Clinical Pharmacology, (2011); 72: 330–337. 2. Leppert, W.; Dihydrocodeine as an opioid analgesic for the treatment of moderate to severe chronic pain; Current Drug Metabolism, (2010); 11: 494–506. 3. Pedersen, L., Borchgrevink, P.C., Breivik, H.P., Fredheim, O.M.S.; A randomized, double-blind, double-dummy comparison of short- and longacting dihydrocodeine in chronic non-malignant pain; Pain, (2014); 155: 881–888. 4. Schmidt, H., Lotsch, J.; Pharmacokinetic–pharmacodynamic modeling of the miotic effects of dihydrocodeine in humans; European Journal of Clinical Pharmacology, (2007); 63: 1045–1054. 5. Al-Asmari, A.I., Anderson, R.A.; The role of dihydrocodeine (DHC) metabolites in dihydrocodeine-related deaths; Journal of Analytical Toxicology, (2010); 34: 476–490. 6. Leppert, W., Mikolajczak, P., Kaminska, E., Szulc, M.; Analgesia and serum assays of controlled-release dihydrocodeine and metabolites in cancer patients with pain; Pharmacological Reports: PR, (2012); 64: 84–93. 7. Tang, W., Liu, X., Qiu, X., Yuan, S., Wang, J., Chen, H.; A simple and sensitive liquid chromatography method for the determination of low dihydrocodeine concentrations in human plasma: its application in Chinese healthy volunteers; Arzneimittelforschung, (2011); 61: 411–416. 8. Klinder, K., Skopp, G., Mattern, R., Aderjan, R.; The detection of dihydrocodeine and its main metabolites in cases of fatal overdose; International Journal of Legal Medicine, (1999); 112: 155–158. 9. Bedford, K.R., White, P.C.; Improved method for the simultaneous determination of morphine, codeine and dihydrocodeine in blood by highperformance liquid chromatography with electrochemical detection; Journal of Chromatography, (1985); 347: 398–404.
UPLC–MS-MS Determination of DHC in Human Plasma 10. Langman, L.J., Korman, E., Stauble, M.E., Boswell, M.V., Baumgartner, R. N., Jortani, S.A.; Therapeutic monitoring of opioids: a sensitive LC–MS-MS method for quantitation of several opioids including hydrocodone and its metabolites; Therapeutic Drug Monitoring, (2013); 35: 352–359. 11. Schaefer, N., Peters, B., Schmidt, P., Ewald, A.H.; Development and validation of two LC–MS-MS methods for the detection and quantification of amphetamines, designer amphetamines, benzoylecgonine, benzodiazepines, opiates, and opioids in urine using turbulent flow chromatography; Analytical and Bioanalytical Chemistry, (2013); 405: 247–258. 12. Cone, E.J., Heltsley, R., Black, D.L., Mitchell, J.M., Lodico, C.P., Flegel, R.R.; Prescription opioids. II. Metabolism and excretion patterns of hydrocodone in urine following controlled single-dose administration; Journal of Analytical Toxicology, (2013); 37: 486–494. 13. Lerch, O., Temme, O., Daldrup, T.; Comprehensive automation of the solid phase extraction gas chromatographic mass spectrometric analysis (SPE-GC/MS) of opioids, cocaine, and metabolites from serum and other matrices; Analytical and Bioanalytical Chemistry, (2014); 406: 4443–4451. 14. Meadway, C., George, S., Braithwaite, R.; A rapid GC–MS method for the determination of dihydrocodeine, codeine, norcodeine, morphine, normorphine and 6-MAM in urine; Forensic Science International, (2002); 127: 136–141. 15. Balikova, M., Maresova, V., Habrdova, V.; Evaluation of urinary dihydrocodeine excretion in human by gas chromatography–mass spectrometry; Journal of Chromatography. B, Biomedical Sciences and Applications, (2001); 752: 179–186.
573 16. Geier, A., Bergemann, D., von Meyer, L.; Evaluation of a solid-phase extraction procedure for the simultaneous determination of morphine, 6-monoacetylmorphine, codeine and dihydrocodeine in plasma and whole blood by GC–MS; International Journal of Legal Medicine, (1996); 109: 80–83. 17. de Villiers, A., Lestremau, F., Szucs, R., Gelebart, S., David, F., Sandra, P.; Evaluation of ultra performance liquid chromatography. Part I. Possibilities and limitations; Journal of Chromatography A, (2006); 1127: 60–69. 18. Qiu, X., Zhao, J., Wang, Z., Xu, Z., Xu, R.A.; Simultaneous determination of bosentan and glimepiride in human plasma by ultra performance liquid chromatography tandem mass spectrometry and its application to a pharmacokinetic study; Journal of Pharmaceutical and Biomedical Analysis, (2014); 95: 207–212. 19. Qiu, X., Wang, Z., Wang, B., Zhan, H., Pan, X., Xu, R.A.; Simultaneous determination of irbesartan and hydrochlorothiazide in human plasma by ultra high performance liquid chromatography tandem mass spectrometry and its application to a bioequivalence study; Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences, (2014); 957: 110–115. 20. Jamalapuram, S., Vuppala, P.K., Abdelazeem, A.H., McCurdy, C.R., Avery, B.A.; Ultra-performance liquid chromatography tandem mass spectrometry method for the determination of AZ66, a sigma receptor ligand, in rat plasma and its application to in vivo pharmacokinetics; Biomedical Chromatography: BMC, (2013); 27: 1034–1040.
Article
UPLC–MS-MS Determination of Dihydrocodeine in Human Plasma and Its Application to a Pharmacokinetic Study Wei-min Zhang1,*, Yan-pei Duan2, Wei Li1, Jian-fei Qiu3, and Zhi-yin Zhang3 1
The First Affiliated Hospital of Henan University of Science and Technology, Luoyang, Henan 471003, PR China, Department of Pharmacy, Xinxiang Central Hospital, Xinxiang, Henan 453000, PR China, and 3Medical College of Henan University of Science and Technology, Luoyang, Henan 471003, PR China
2
*Author to whom correspondence should be addressed. Email: [email protected] Received 20 December 2014; Revised 23 October 2015
Abstract A sensitive and rapid ultra performance liquid chromatography tandem mass spectrometry (UPLC– MS-MS) method was developed to determine dihydrocodeine (DHC) in human plasma using diazepam as the internal standard (IS). Sample preparation was accomplished through a liquid–liquid extraction procedure with ethyl acetate. The analyte and IS were separated on an Acquity UPLC BEH C18 column (2.1 × 50 mm, 1.7 μm) with the mobile phase of acetonitrile and 1% formic acid in water with gradient elution at a flow rate of 0.4 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer equipped with positive-ion electrospray ionization by multiple reaction monitoring (MRM) of the transitions at m/z 302.3 → 199.2 for DHC and m/z 285.1 → 193.1 for IS. The linearity of this method was found to be within the concentration range of 0.5–100 ng/mL with a lower limit of quantification of 0.5 ng/mL. The overall run time was 4.0 min. The method herein described was superior to previous methods and was successfully applied to the pharmacokinetic study of DHC in healthy Chinese volunteers after oral administration.
Introduction Dihydrocodeine (DHC, Figure 1) is a semi-synthetic opioid, licensed in most countries to treat moderate to severe pain (1, 2). It is a so-called “weak” opioid that can be used if nonopioid analgesics such as paracetamol and nonsteroidal anti-inflammatory drugs provide insufficient pain control (2, 3). Bioavailability of DHC after oral administration is ∼20%, which indicates that its analgesic efficacy after oral administration is slightly stronger than that of codeine (bioavailability after oral administration approximately equals 30–40%) (4). After DHC oral administration, the maximal serum concentration appears after 1.7 h, plasma half-life is 3.5–5.5 h and analgesia period is 4 h. However, there is little data available on DHC pharmacokinetics. Several methods have been published for the determination of DHC in biological matrices, including high-performance liquid chromatography (HPLC) (5–9), high-performance liquid chromatography tandem mass spectrometry (HPLC–MS-MS) (10–12) and gas chromatography–mass spectrometry (GC–MS) (13–16). However, these
methods have their own disadvantages, such as the lack of sensitivity, complex analyzing processes, long time requirement for preparation of samples and long chromatographic run times, which are not preferable when dealing with a large number of samples. Moreover, no publication has described the quantitative analysis of DHC in the biological fluid using ultra performance liquid chromatography tandem mass spectrometry (UPLC–MS-MS). Therefore, a novel, simple and rapid method for the measurement of DHC concentration in human plasma sample is still required for therapeutic drug monitoring. UPLC–MS-MS has been considered as a faster and more efficient analytical tool compared with current chromatography (17–19). In this study, a UPLC–MS-MS method for the determination of DHC using diazepam as an internal standard (IS) was developed. This new method has been fully validated in terms of selectivity, linearity, lower limit of quantification (LLOQ), accuracy, precision, stability, matrix effect and recovery. It has been successfully applied in a pharmacokinetic study conducted in Chinese healthy male volunteers.
© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
569
570
Zhang et al. for dilution. All stock solutions, working solutions, calibration standards and QCs were immediately stored at −20°C.
Sample preparation
Figure 1. The chemical structures of DHC and IS in this study: (A) DHC and (B) diazepam (IS).
Experimental Chemical materials DHC and diazepam (IS) were obtained from Sigma (St. Louis, MO, USA). Acetonitrile and methanol were of HPLC grade and purchased from Merck Company (Darmstadt, Germany). HPLC grade water was obtained using a Milli Q system (Millipore, Bedford, USA).
UPLC–MS-MS conditions Liquid chromatography was performed on an Acquity ultra performance liquid chromatography (UPLC) unit (Waters Corp., Milford, MA, USA) with an Acquity BEH C18 column (2.1 × 50 mm, 1.7 μm particle size) and inline 0.2 μm stainless steel frit filter (Waters Corp.). A gradient elution program was conducted for chromatographic separation with mobile phase A (acetonitrile), and mobile phase B (0.1% formic acid in water) as follows: 0–0.5 min (20–75% A), 0.5–2.0 min (75–75% A), 2.0–2.1 min (75–20% A) and 2.1– 4.0 min (20–20% A). The flow rate was 0.4 mL/min. The overall run time was 4.0 min. A XEVO TQD triple quadrupole mass spectrometer equipped with an electro-spray ionization (ESI) source was used for mass spectrometric detection. The detection was operated in the multiple reaction monitoring (MRM) mode under unit mass resolution in the mass analyzers. The MRM transitions were m/z 302.3 → 199.2 and m/z 285.1 → 193.1 for DHC and IS, respectively. After optimization, the source parameters were set as follows: curtain gas, 35 psig; nebulizer gas, 50 psig; turbo gas, 60 psig; ion spray voltage, 4.0 kV and temperature, 500°C. Data acquisition and processing were performed using Analyst Software (Waters Corp.).
Standard solutions, calibration standards and quality control sample The stock solution of DHC that was used to make the calibration standards and quality control (QC) samples was prepared by dissolving 10 mg in 10 mL methanol to obtain a concentration of 1.0 mg/mL. The stock solution was further diluted with methanol to obtain working solutions at several concentration levels. Calibration standards and QC samples in plasma were prepared by diluting the corresponding working solutions with blank human plasma. Final concentrations of the calibration standards were 0.5, 1, 2, 5, 10, 20, 50 and 100 ng/mL for DHC in human plasma. The concentrations of QC samples in plasma were 1, 8 and 80 ng/mL for DHC. IS stock solution was made at an initial concentration of 1 mg/mL. The IS working solution (200 ng/mL) was made from the stock solution using methanol
Before analysis, the plasma samples were thawed to ambient temperature. In a 1.5-mL centrifuge tube, an aliquot of 20 µL of the IS working solution (200 ng/mL) and 100 µL of NaOH (1 mol/L) were added to 200 µL of the collected plasma sample followed by the addition of 1.0 mL ethyl acetate. The tubes were vortex mixed for 1.0 min. After centrifugation at 8,000 × g for 10 min, the supernatant organic layer was transferred into a 1.5-mL centrifuge tube and dried under nitrogen stream at 40°C. The dried residue was reconstituted in 80 µL of acetonitrile and water (1 : 1, v/v), and a 3-µL aliquot of this was injected into UPLC–MS-MS system for the analysis.
Method validation Before using this method to determinate DHC in human plasma, the method was fully validated for specificity, linearity, precision, accuracy, recovery, matrix effect and stability according the United States Food and Drug Administration bioanalytical method validation guidances (20). Specificity was determined by analysis of blank human plasma samples from six different volunteers, every blank sample was handled by the procedure described in the “Sample preparation” section and confirmed that endogenous substances did not have the possible interference with the analyte and the IS. To evaluate the linearity, calibration standards of nine concentrations of DHC (0.5–100 ng/mL) were separately extracted and assayed on three separate days. The linearity for DHC was investigated by weighted (1/x 2) least-squares linear regression of peak area ratios against concentrations. The sensitivity of the method was determined by quantifying the LLOQ. The LLOQ was defined as the lowest acceptable point in the calibration curve, which was determined at an acceptable precision and accuracy. To determine the matrix effect, six different blank human samples were utilized to prepare QC samples and used for assessing the lot-to-lot matrix effect. Matrix effect was evaluated at three QC levels by comparing the peak areas of analytes obtained from plasma samples spiked with analytes after extraction to those of the pure standard solutions at the same concentrations. The matrix effect of IS was evaluated at the working concentration (50 ng/mL) in the same manner. The precision and accuracy of the method were assessed by determination of QC samples in plasma at different concentrations (1, 8 and 80 ng/mL) on three separate days. Precision was expressed as % relative standard deviation (RSD) and accuracy was expressed as % relative error (RE) between the measured and nominal value. The precision for QC samples was within 15%, and accuracy was between −15 and 15%. Extraction recovery experiments, which showed an ability to extract the analyte from the test biological samples, were evaluated by comparing the peak areas obtained from extracted QC samples with nonprocessed standard solutions at three concentrations at the same concentration. Recovery of IS was determined at the working concentration (50 ng/mL) similarly. The stability in human plasma was tested by analyzing five replicates of plasma samples at three concentration levels (1, 8 and 80 ng/mL) under different conditions. The short-term stability was determined after the exposure of the spiked samples at ambient temperature for 3 h, and the ready-to-inject samples (after extraction) in the autosampler at 4°C for 24 h. The freeze–thaw stability was evaluated
571
UPLC–MS-MS Determination of DHC in Human Plasma after three complete freeze–thaw cycles (−20 to 25°C) on consecutive days. The long-term stability was assessed after storage of the standard spiked plasma samples at −20°C for 28 days. Samples were considered to be stable if assay values were within the acceptable limits of accuracy (RE % ≤ ±15%) and precision (RSD % ≤ 15%).
Application to a pharmacokinetic study The present method was applied to a pharmacokinetic study after an oral administration of 20 mg DHC tablets to Chinese male volunteers. The clinical protocol was approved by Medical Ethics Committee of Henan University of Science and Technology prior to the study. Twenty volunteers (aged 21–28 years, weighing 50–70 kg, and height 156– 180 cm) signed the informed consent form and participated in the study according to the principles of the Declaration of Helsinki. The volunteers who submitted the agreements to attend this project were medically examined for the pharmacokinetic study of DHC. The subjects were required to abstain from taking any other drug for 7 days prior to the start of test. They were also demanded to abstain from smoking or drinking alcohol for 24 h before the beginning of the study until its end. All volunteers received an oral dose of 20 mg DHC with 200 mL water. Blood samples (3 mL) were collected into heparinized tubes before and 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 4,000 × g for 8 min, and the plasma was separated and kept frozen at −20°C until analysis.
Data analysis Plasma concentration versus time profiles were analyzed using DAS (Drug and Statistics Software; version 2.0, Henan University of Science and Technology, China) to estimate the type of compartment model and pharmacokinetic parameters. Data were expressed as mean ± SD.
Results Specificity and matrix effect UPLC–MS-MS chromatogram of the analytes in human plasma samples is shown in Figure 2. The retention times of DHC and IS are 1.17 and 1.38 min, respectively. Compared with chromatogram of blank blood sample, no interference of endogenous peaks was observed. The matrix effects for DHC at concentrations of 1, 8 and 80 ng/mL were measured to be 108.4, 110.5 and 93.6% (n = 6), respectively. The ME for IS (50 ng/mL) was 97.3% (n = 6). No apparent matrix
effect was found to affect the determination of DHC and IS in plasma. As a result, the matrix effect from plasma was negligible in this method.
Linearity and sensitivity The peak area ratios of DHC/IS versus the nominal concentrations of DHC showed a good linear relationship over the concentration ranges of 0.5–100 ng/mL in human plasma. A typical regression equation for the calibration curve resulted in an equation of the line of: y = 0.0174x – 0.00792, where y represents the peak area ratios of DHC to the IS and x represents plasma concentrations of the analyte. The LLOQ in human plasma was 0.5 ng/mL with the RSD and RE of 9.5 and 7.3%, respectively.
Precision, accuracy and recovery The intra- and interday precision and accuracy of the method were determined from the analysis of QC samples at three different concentrations (1, 8 and 80 ng/mL) for each biological matrix. The method was reliable and reproducible because RSD % was below 10% and RE % was between −10.5 and 9.7% for all the investigated concentrations of DHC in human plasma. The recovery was calculated by comparing the mean peak areas of the analyte spiked before extraction divided by the areas of analyte samples spiked after extraction and multiplied by 100%. The recovery in plasma ranged from 75.7 to 85.8% for DHC. The recovery of IS (50 ng/mL) in plasma was 83.1%. Assay performance data are presented in Table I. The above results demonstrated that the values were within the acceptable range, and the method was accurate and precise.
Stability Stability tests were performed at the low, medium and high QC samples with five determinations for each under different storage conditions. The RSDs of the mean test responses were within 15% in all stability tests. There was no effect on the quantitation for plasma samples kept at ambient temperature for 3 h and at 4°C for 24 h. No significant degradation was observed when samples of DHC were taken through three freeze (−20°C)–thaw (ambient temperature) cycles. As a result, DHC in samples were stable at −20°C for 28 days.
Application of the method in a pharmacokinetic study The method described above was successfully applied to determine the concentration of DHC in human plasma. After an oral administration of 20 mg DHC tablets, the main pharmacokinetic parameters of DHC
Figure 2. Representative chromatograms of DHC and IS in human plasma samples. (A) A blank plasma sample; (B) a blank plasma sample spiked with DHC and IS and (C) a plasma sample from a person 0.5 h after an oral administration of 20 mg. This figure is available in black and white in print and in color at JCS online.
572
Zhang et al.
Table I. Precision, Accuracy and Recovery for DHC of QC Samples in Human Plasma (n = 6)
Table II. Pharmacokinetic Parameters of DHC After Oral Administration of 20 mg to 20 Chinese Healthy Male Volunteers
Concentration (ng/mL)
RSD%
Parameters
Mean ± SD
Intraday
Interday
Intraday
Interday
0.5 1 8 80
7.5 8.4 4.7 6.3
8.8 9.3 7.8 5.5
9.7 −10.5 7.4 −6.3
−11.3 8.1 −4.8 9.7
t1/2 (h) Cmax (ng/mL) Tmax (h) AUC0→14 (ng/mL h) AUC0→∞ (ng/mL h)
3.146 ± 0.356 60.089 ± 8.318 0.844 ± 0.221 285.394 ± 28.606 301.643 ± 31.051
RE%
Recovery (%) 77.5 80.4 75.7 84.8
mobile phases, acetonitrile–water (containing 0.1% formic acid) was found to be optimal for this study, which provided symmetric peak shapes of the analyte and IS. The total time required for the chromatographic run was 4.0 min. The addition of 0.1% formic acid helped to obtain improved signals.
Conclusion
Figure 3. Plasma concentration versus time curves of DHC after a single oral administration of 20 mg to 20 Chinese healthy male volunteers.
for 20 volunteers were estimated. The mean plasma concentration– time curve of DHC is displayed in Figure 3, and the main pharmacokinetic parameters of DHC were calculated and are summarized in Table II. Compared with recently published articles describing the pharmacokinetic profiles of DHC in healthy volunteers, the pharmacokinetic parameters of DHC obtained in the study were generally similar (7).
Discussion In this study, different sample treatment approaches were investigated to achieve a simple and inexpensive extraction procedure. Protein precipitation extraction was initially tried, but low DHC concentration was not detectable during our method development. Then, a liquid– liquid extraction (LLE) technique was investigated and found to be more sensitive. LLE methods using a mixture of organic solvents have been investigated for the extraction of DHC from biological samples. In this study, different organic solvents including diethyl ether, chloroform and ethyl acetate were investigated. Among them, ethyl acetate was found to be optimal solvent for extraction yielding satisfactory results in terms of both sample cleaning and the analyte extraction recovery. Moreover, the boiling point of ethyl acetate is low, so it was evaporated to dryness more quickly. Hence, ethyl acetate was chosen as the extraction solvent. This one-step extraction with ethyl acetate is cheaper than the reported method, which makes the method suitable for low-cost clinical study (16). Further optimization in chromatography conditions was performed to test different mobile phases. In our assessment of different
An UPLC–MS-MS method for the determination of DHC in human plasma was developed and validated. To the best of our knowledge, this is the first report of the determination of the DHC level in human plasma using an UPLC–MS-MS method. Compared with the analytical methods reported in the literature, the method offered superior sample preparation with LLE with organic solvent to precipitation of plasma protein and a shorter run time of 4.0 min. The method meets the requirement of high sample throughput in bioanalysis and has been successfully applied to the pharmacokinetic study of DHC tablets in healthy volunteers.
References 1. Zamparutti, G., Schifano, F., Corkery, J.M., Oyefeso, A., Ghodse, A.H.; Deaths of opiate/opioid misusers involving dihydrocodeine, UK, 1997– 2007; British Journal of Clinical Pharmacology, (2011); 72: 330–337. 2. Leppert, W.; Dihydrocodeine as an opioid analgesic for the treatment of moderate to severe chronic pain; Current Drug Metabolism, (2010); 11: 494–506. 3. Pedersen, L., Borchgrevink, P.C., Breivik, H.P., Fredheim, O.M.S.; A randomized, double-blind, double-dummy comparison of short- and longacting dihydrocodeine in chronic non-malignant pain; Pain, (2014); 155: 881–888. 4. Schmidt, H., Lotsch, J.; Pharmacokinetic–pharmacodynamic modeling of the miotic effects of dihydrocodeine in humans; European Journal of Clinical Pharmacology, (2007); 63: 1045–1054. 5. Al-Asmari, A.I., Anderson, R.A.; The role of dihydrocodeine (DHC) metabolites in dihydrocodeine-related deaths; Journal of Analytical Toxicology, (2010); 34: 476–490. 6. Leppert, W., Mikolajczak, P., Kaminska, E., Szulc, M.; Analgesia and serum assays of controlled-release dihydrocodeine and metabolites in cancer patients with pain; Pharmacological Reports: PR, (2012); 64: 84–93. 7. Tang, W., Liu, X., Qiu, X., Yuan, S., Wang, J., Chen, H.; A simple and sensitive liquid chromatography method for the determination of low dihydrocodeine concentrations in human plasma: its application in Chinese healthy volunteers; Arzneimittelforschung, (2011); 61: 411–416. 8. Klinder, K., Skopp, G., Mattern, R., Aderjan, R.; The detection of dihydrocodeine and its main metabolites in cases of fatal overdose; International Journal of Legal Medicine, (1999); 112: 155–158. 9. Bedford, K.R., White, P.C.; Improved method for the simultaneous determination of morphine, codeine and dihydrocodeine in blood by highperformance liquid chromatography with electrochemical detection; Journal of Chromatography, (1985); 347: 398–404.
UPLC–MS-MS Determination of DHC in Human Plasma 10. Langman, L.J., Korman, E., Stauble, M.E., Boswell, M.V., Baumgartner, R. N., Jortani, S.A.; Therapeutic monitoring of opioids: a sensitive LC–MS-MS method for quantitation of several opioids including hydrocodone and its metabolites; Therapeutic Drug Monitoring, (2013); 35: 352–359. 11. Schaefer, N., Peters, B., Schmidt, P., Ewald, A.H.; Development and validation of two LC–MS-MS methods for the detection and quantification of amphetamines, designer amphetamines, benzoylecgonine, benzodiazepines, opiates, and opioids in urine using turbulent flow chromatography; Analytical and Bioanalytical Chemistry, (2013); 405: 247–258. 12. Cone, E.J., Heltsley, R., Black, D.L., Mitchell, J.M., Lodico, C.P., Flegel, R.R.; Prescription opioids. II. Metabolism and excretion patterns of hydrocodone in urine following controlled single-dose administration; Journal of Analytical Toxicology, (2013); 37: 486–494. 13. Lerch, O., Temme, O., Daldrup, T.; Comprehensive automation of the solid phase extraction gas chromatographic mass spectrometric analysis (SPE-GC/MS) of opioids, cocaine, and metabolites from serum and other matrices; Analytical and Bioanalytical Chemistry, (2014); 406: 4443–4451. 14. Meadway, C., George, S., Braithwaite, R.; A rapid GC–MS method for the determination of dihydrocodeine, codeine, norcodeine, morphine, normorphine and 6-MAM in urine; Forensic Science International, (2002); 127: 136–141. 15. Balikova, M., Maresova, V., Habrdova, V.; Evaluation of urinary dihydrocodeine excretion in human by gas chromatography–mass spectrometry; Journal of Chromatography. B, Biomedical Sciences and Applications, (2001); 752: 179–186.
573 16. Geier, A., Bergemann, D., von Meyer, L.; Evaluation of a solid-phase extraction procedure for the simultaneous determination of morphine, 6-monoacetylmorphine, codeine and dihydrocodeine in plasma and whole blood by GC–MS; International Journal of Legal Medicine, (1996); 109: 80–83. 17. de Villiers, A., Lestremau, F., Szucs, R., Gelebart, S., David, F., Sandra, P.; Evaluation of ultra performance liquid chromatography. Part I. Possibilities and limitations; Journal of Chromatography A, (2006); 1127: 60–69. 18. Qiu, X., Zhao, J., Wang, Z., Xu, Z., Xu, R.A.; Simultaneous determination of bosentan and glimepiride in human plasma by ultra performance liquid chromatography tandem mass spectrometry and its application to a pharmacokinetic study; Journal of Pharmaceutical and Biomedical Analysis, (2014); 95: 207–212. 19. Qiu, X., Wang, Z., Wang, B., Zhan, H., Pan, X., Xu, R.A.; Simultaneous determination of irbesartan and hydrochlorothiazide in human plasma by ultra high performance liquid chromatography tandem mass spectrometry and its application to a bioequivalence study; Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences, (2014); 957: 110–115. 20. Jamalapuram, S., Vuppala, P.K., Abdelazeem, A.H., McCurdy, C.R., Avery, B.A.; Ultra-performance liquid chromatography tandem mass spectrometry method for the determination of AZ66, a sigma receptor ligand, in rat plasma and its application to in vivo pharmacokinetics; Biomedical Chromatography: BMC, (2013); 27: 1034–1040.
