Journal of Chromatographic Science 2014;52:1217– 1223 doi:10.1093/chromsci/bmt200 Advance Access publication January 20, 2014
Article
Determination of Paroxetine in Blood and Urine Using Micellar Liquid Chromatography with Electrochemical Detection Nitasha Agrawal1, Sergio Marco-Peiro´2, Josep Esteve-Romero2, Abhilasha Durgbanshi1, Devasish Bose1, Juan Peris-Vicente2* and Samuel Carda-Broch2 1
Department of Criminology and Forensic Sciences, Dr. H.S. Gour University, Sagar, India and 2A`rea de Quı´ mica Analı´ tica, Universitat Jaume I, Castello´ 12071, Spain
*Author to whom correspondence should be addressed. Email: [email protected] Received 30 April 2013; revised 23 September 2013
Paroxetine is a potent selective serotonin reuptake inhibitor used for the treatment of depression and related mood disorders. A micellar liquid chromatographic method was developed for the determination of paroxetine in serum and urine. Detection of paroxetine was carried out using a C18 column and a mobile phase of 0.15 M sodium dodecyl sulfate, 6% 1-pentanol at pH 3 (buffer salt 0.01 M NaH2PO4) running under isocratic mode at 1.0 mL/min and electrochemical detection at 0.8 V. The analyte was eluted without interferences in 0.9999; 0.5 –5 mg/mL range), accuracy (88–97.5%, recovery), repeatability (RSD < 0.54%), intermediate precision (RSD < 0.54%), limit of detection and quantification (0.001 and 0.005 mg/mL, respectively) and robustness (RSD < 3.63%). Developed method was successfully applied to real blood and urine samples as well as in spiked serum and urine samples. The developed method was specific, rapid, precise, reliable, accurate, inexpensive and then suitable for routine analysis of paroxetine in monitorized samples.
Introduction In the last few years, the prescription of antidepressants has increased many folds. The new drugs that enhance serotoninergic neurotransmission by potent and selective inhibition of serotonin are highest among the prescribed antidepressants, they are known as selective serotonin reuptake inhibitors (SSRI) (1). Paroxetine, (2)-trans-4-(4-fluorophenyl)-3-(3,4-methylenedioxy-phenoxymethyl) piperidine (Table I), is one of the SSRIs used widely as an antidepressant drug alone or in combination with other drugs (2). Paroxetine can be compared with the tricyclic antidepressants in their clinical efficacy; however, it is safer and has greater acceptance by the patients. Paroxetine is devoid of sedative effect and remarkably safe in overdose. It takes 5.2 h to reach the peak, with extended half-life (21 h) that allowed the introduction of formulations for once daily dosing. The above-mentioned qualities of paroxetine made the drug as the most widely prescribed antidepressant (3). It is used in the treatment of clinical depression, obsessive–compulsive disorder and panic disorder. In a survey performed by NDC Health (Atlanta, GA, USA) in 2011, paroxetine was one of the top 150 prescriptions for 2011 by number of US prescriptions dispensed (http://www.rxlist.com/script/main/ hp.asp).
Once administered, paroxetine is mainly oxidized to an unstable catechol followed by further methylation, and the major metabolite is conjugated rapidly to glucuronide and sulfate ethers in animals and humans. This major metabolite has not been found to inhibit serotonin reuptake significantly. Paroxetine and its metabolites in human plasma, serum or in tablets have been quantified by high-performance liquid chromatography (HPLC) (4). During the literature survey, it was also found that along with its benefits, paroxetine do possess some serious health risks. Patients with depression are at a high risk of suicide attempts (5). The European Medicines Agency’s Committee for Medicinal Products for Human Use (CHMP) published a letter for patients, prescribers and parents that paroxetine should not be prescribed to children. CHMP gave a warning to prescribers recommending close monitoring of adult patients at high risk of suicidal behavior and/or suicidal thoughts. The drug is often related with the withdrawal symptoms on stopping treatment abruptly and, therefore, it is recommended to gradually reduce the dose over several weeks or months if decision of withdrawal is made (6). A survey carried out by Food and Drug Administration in 2004 on children prescribed with paroxetine showed 2.7-fold raise in suicide behavior and ideation when compared with placebo. The trend for increased suicidality was observed in both trials for depression and for anxiety disorders (7). Several methods have been published for the determination of paroxetine in plasma, including reversed phase HPLC with UV (8, 9), fluorescence (with or without derivatization using dansylchloride) (10 –12), MS (13 –15), diode array detection (16, 17) or gas chromatography with MS detection (18 –20), and applied therapeutic drug level monitoring or bioequivalence studies. Only a few methods have been published for the simultaneous determination of paroxetine and its metabolites in plasma, including HPLC with UV (4, 8), and applied to pharmacokinetic, therapeutic drug level monitoring and toxicological screening or forensic cases. It was also noted that there were no papers published describing the use of micellar liquid chromatography (MLC) for the analysis of paroxetine using electrochemical detection (ECD). MLC is one of the most frequently used direct injection chromatographic technique which uses a surfactant at a concentration above the critical micellar concentration as mobile phase, presenting an alternative to conventional HPLC. The simultaneous elution of hydrophobic and hydrophilic analytes is possible without needing a gradient elution, and direct injection of physiological samples becomes feasible due to the solubility of proteins in the micelles, avoiding long and tedious extraction steps. MLC
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Table I. Structure and Physical Properties of Paroxetine Compound Paroxetine
Structure
pKa
Log Po/w
9.9
3.95
has been proved to be a useful technique in the determination of diverse group of compounds. In addition, the stable and reproducible behavior of micellar mobile phases allows accurate prediction of solute retention with a model that can also be used to optimize the separation of mixtures. MLC technique has been successfully used in the determination of large number of drugs in pharmaceutical preparations, biological fluids (serum and urine) that can be injected directly into the chromatographic system. Sodium dodecyl sulfate (SDS) is a widely used anionic surfactant in micellar media given its solubility in water, its low critic micellar concentration, low cost, and because it is easy to remove from the chromatographic system. The use of MLC expedites the experimental procedure, reducing the analysis time and the cost of the methodology (21–25). The purpose of the present work was to develop a rapid, simple and selective MLC procedure for the screening of paroxetine in urine, serum and physiological samples, using C18 column and ECD. The determination of paroxetine was simplified by direct injection of serum and urine samples in the MLC system, and the use of ECD reduces the protein band, thus increasing the selectivity. The proposed methodology was validated following the guidelines of the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) in serum in terms of specificity, linearity, accuracy, repeatability, intermediate precision, sensitivity (limits of detection and quantification, LOD and LOQ) and robustness (26). The suitability of the method to routine analysis of blood and urine samples of paroxetine takers was also considered.
Experimental Materials and reagents Standard paroxetine ( purity .99.99%) was kindly gifted from Laboratorio STADA, S.L. (Sant Just Desvern, Barcelona, Spain). SDS salt ( purity .99%) and potassium chloride ( purity .99%) was purchased from Merck (Darmstadt, Germany). Sodium dihydrogen phosphate ( purity .99%) and 37% HCl (from Panreac, Barcelona, Spain) and methanol, 1-propanol, 1-butanol and n-amyl alcohol (HPLC grade) were purchased from Scharlau (Barcelona, Spain). The serum was gifted by Lifeline pathology, Sagar, India and Hospital Provincial, Castellon, Spain. Urine was collected from healthy drug-free donors. Ultrapure water used throughout the study was obtained from distilled water by an Ultrapure Water device from Millipore S.A.S. (Molsheim, France). 1218 Agrawal et al.
Apparatus and instrumentation An Agilent Technologies Chromatograph (Model HP1100, Palo Alto, CA, USA), equipped with a quaternary pump, an autosampler and electrochemical (ECD, Model HP 1049A) detectors were used. Monitoring was performed at 800 mV with electrochemical detector with an Ag/AgCl as reference and glassy carbon as working electrode. A Kromasil C18 column (Scharlab, 5 mm particle size, 120 4.6 mm i.d.) was employed for the analytical separation. Flow rate and the injection volume were 1.0 mL/min and 20 mL, respectively. The dead time was determined as the mean value of the first significant deviation of the baseline in the chromatograms of the analytes. Signal was acquired by a PC connected to the chromatograph, through an HP Chemstation (Rev. B.03.01). An ultrasonic bath (model Ultrasons-H, Selecta, Abrera, Spain) was used to solve the standards and the pH of the solutions was measured with a Crison GLP 22 (Barcelona) potentiometer equipped with a combined Ag/AgCl/glass electrode. The analytical balance used was a Mettler-Toledo AX105 Delta-Range (Greifensee, Switzerland).
Sample preparation The drug sample was first weighed and then dissolved in methanol. The solutions were injected into the chromatograph without any pretreatment other than filtration, which was carried out directly into the autosampler vials through 0.45 mm nylon membranes. Both the serum and urine samples were first diluted in a ratio of 1:5 with the mobile phase before injection.
Mobile phase preparation Micellar mobile phases were prepared by weighing an exact amount ofSDS, sodium dihydrogen phosphate and KCl and dissolving them in water, adjusting the pH to 3 by adding drops of an HCl 6 M solution to reach the desired pH. A suitable volume of alcohol (i.e., 1-propanol, 1-butanol or 1-pentanol), depending on the percentage was added to the mobile phase as organic modifier. The usefulness of micellar mobile solvents and phases lies in the direct injection of physiological fluids as the monomers and micelles tend to bind proteins competitively; thereby releasing protein bound drugs which are free to partition into the stationary phase. Finally, the proteins, rather than precipitating into the column are solubilized and swept away harmlessly, eluting with or shortly after the solvent front. Due to the use of salt solutions as a mobile phase, special careful considerations are required to prevent the precipitation of SDS which would seriously damage the chromatographic system (27).
Results Method development and optimization Appropriate choice of stationary and mobile phase is very important for developing methods in micellar liquid chromatography. Selection of pH along with the appropriate oxidation potential is the first parameter to be studied when a micellar
procedure is developed using ECD. The amount of modifier in % plays a very significant role. Selection of oxidation potential Paroxetine is the only drug among SSRIs which is electrochemically active, which automatically filters it from other prescribed drugs of similar nature or structure. In order to establish the optimum oxidation potential, paroxetine was oxidized using 200 –900 mV, in steps of 100 up to 600 mV and then in steps of 50 mV. On the basis of the data obtained, 800 mV was selected for the analysis Figure 1. Selection of pH The pH variation of the mobile phase will affect the retention time of weak acids. Then pH must be fixed by the use of a buffer. The working pH range of the column is 1.5 –9.5, but the use of basic mobile phase causes the degradation of the C18 coating of the stationary phase, so that only acidic or neutral pH was considered (28). As pKa of paroxetine is 9.9 (Table I), at these pH, the analyte is under its neutral form. The analysis of paroxetine was carried out at pH 3, 5 and 7. The areas of the chromatographic peak of paroxetine were similar in the three cases, but in ECD, the micellar media get somewhat more oxidized at pH 7, resulting in a high background noise (baseline width: 14 mA for pH 7 and 6 mA for pH 3). Thus based on the value of pKa, structure and data obtained, it was decided to use pH 3 for further studies. Selection of organic modifier The partition coefficient of paroxetine (log Po/w ¼ 3.95; see Table I) suggests that the substance is highly hydrophobic. These kinds of compounds are not eluted in feasible times using pure aqueous micellar mobile phases and therefore need the addition of an organic modifier. Short-chain alcohols such as 1-propanol, 1-butanol and 1-pentanol could be appropriately
added to the mobile phase for achieving a higher elution strength that increases according to the length of their carbon chain (29). The concentration and the nature of the short-chain alcohol are critical in the separation. The use of higher concentrations of short-chain alcohol is not recommended in MLC. The alcohol tends to incorporate in the micelle structure (competing with SDS by the position of the micelle). This reduces the stability of the micelle and favors its destruction. Therefore, the number of micelles is lower than the expected and not reproducible, disturbing the analysis. The affinity of the alcohol with the micelle increases with the hydrophobicity of the alcohol. Therefore, the maximum tolerable concentration is increased when the carbon chain is shorter. Maximum tolerated concentrations for each short-chain alcohol are the values indicated in Table II (30). To select the organic modifier, a serum sample free of paroxetine and a blank serum sample spiked with 2.5 mg/mL were analyzed (by six replicates). Three mobile phases with maximum concentration of SDS (0.15 M) and a high concentration of alcohol (12% 1-propanol, 7% 1-butanol and 6% 1-pentanol) were tested. The retention time, retention factor (k0 ), efficiency (N) and asymmetry (B/A) of the chromatographic peak corresponding to paroxetine were determined for each tested mobile phase, and the results are shown in Table II. Effectively, elution time was lower and efficiency higher using short-chain alcohols at increasing molecular weight. Finally, the optimum mobile phase selected was based on the minimum analysis time, high resolution and adequate peak shape. The selected mobile phase was 0.15 M SDS– 6% 1-pentanol at pH 3. Under these conditions, the chromatogram sample serum free of paroxetine shows a wide front (to 4 min), but no other peak was observed at higher elution times (Fig. 2A). In the chromatogram from spiked serum sample, the chromatographic peak of paroxetine appears at 11.00 + 0.04 min without interferences and shows an adequate shape (Fig. 2C). The retention time was low but enough far from the dead time (4 min), which is useful for routine analysis.
Validation The developed method was validated for specificity, accuracy, linearity, LOD, LOQ, repeatability, intermediate precision and robustness. The method was developed in serum and urine. As results are similar for both cases, the values for serum are shown. Specificity The specificity is the ability to assess unequivocally the analyte in the presence of other drugs which can be prescribed together or other compounds of the matrices, especially proteins. In the
Table II. Chromatographic Parameter of the Peak Corresponding to Paroxetine at Different Organic Modifier Added to a 0.15 M SDS Aqueous Mobile Phase Buffered at pH 3
Figure 1. Chromatograms obtained by analyzing a standard solution 5 mg/mL of paroxetine by MLC-ECD at several oxidation potential.
Organic modifier (amount)
Retention time (min)
k0 (retention factor)
N (efficiency)
B/A (asymmetry)
1-Propanol (12%) 1-Butanol (7%) 1-Pentanol (6%)
31.6 + 0.3 22.46 + 0.05 11.00 + 0.04
32.26 22.64 10.57
1289 + 3 1546.5 + 0.9 1790.8 + 0.5
0.916 + 0.007 1.124 + 0.008 0.974 + 0.005
n ¼ 6.
Determination of Paroxetine in Blood 1219
Figure 2. Obtained chromatogram using 0.15 M SDS, 6% 1-pentanol at pH 3, 1.0 mL/min and ECD 0.8 V for injection of (A) blank serum, (B) blank urine, (C) serum spiked with 2.5 mg/mL paroxetine and (D) urine spiked with 2.5 mg/mL.
case of chromatographic analysis, it should be assessed that there is not compounds in the matrices with the same retention time. As has been previously indicated (Selection of oxidation potential section), SSRIs (except paroxetine) are not electrochemically active and then could not interfere with the quantification of the analyte. On the other hand, micellar mobile phases provoke the solubilization of proteins and their elution at the dead time (,4 min) (27). Paroxetine-free serum and urine samples were selected as controls and processed directly in the chromatographic system. They were then analyzed to determine the presence in serum (Fig. 2A) and urine (Fig. 2B) of endogenous components which can overlap with the analyte. Then they were compared with spiked samples with 2.5 mg/mL to evaluate the specificity of the 1220 Agrawal et al.
method. No interference from endogenous compounds was found in serum (Fig. 2C) neither in urine (Fig. 2D). Linearity and limit of detection For linearity study, paroxetine was injected at different concentrations after dilution of the stock solution with matrix samples free of paroxetine. Under the selected chromatographic conditions, the linear range of the signal response for the drug was studied in the concentration ranging from 0.05 to 5 mg/mL at nine different concentration levels, analysis of each calibration level was replicated six times. The slope, intercept and regression coefficient (r 2) were determined by plotting the area of the chromatographic peak of the paroxetine versus the concentration using the least-square linear regression analysis method. To study the variability of the calibration parameters, five curves
were constructed by 5 days over a 2-month period using different sets of standard, and the average values were shown. The adjusted equations and regression coefficients for paroxetine in serum were: A ða.u.Þ ¼ ð55:977 + 0:008Þ ½ paroxetine ðmg/mL) þ ð0:4 + 0:5Þr 2 . 0:9999 The LOD and LOQ for the drug were determined with the 3 s criterion and 10 s criterion, respectively (as indicated in ICH guidelines), using a series of 10 solutions containing low concentrations (0.05 –5 mg/mL) of the drug. The results were based not only on the standard deviation of the response but also on the slope of a specific calibration curve containing the analyte. Under these conditions, the LOD and LOQ were 0.001 and 0.05 mg/mL, respectively. Repeatability and intermediate precision Repeatability and intermediate precision of the suggested methodology were determined using blank matrix samples spiked at three concentration levels: 1, 2.5 and 5 mg/mL. Repeatability was determined by analyzing six replicated of each concentration in the same day and calculating RSD of the areas of the chromatographic peak corresponding to paroxetine (intraday precision), whereas intermediate precision correspond to the average of five measurements of the intraday values over a 3-month period (interday precision). The results for 1, 2.5 and 5 mg/mL were: repeatability 0.13, 0.54 and 0.35%, respectively, and intermediate precision 0.17, 0.16 and 0.54%, respectively. Robustness The robustness was evaluated by analyzing a blank serum spiked with 1 mg/mL of paroxetine standard (n ¼ 6) by making slight deliberate changes to the following parameters: SDS concentration, 1-pentanol amount, pH, applied voltage, flow rate and injection volume. The variation of sensitivity (area of chromatographic peak), retention time, efficiency and asymmetry was measured as RSD, and the results are shown in Table III. The variations in all the studied chromatographic parameters were: area of chromatographic peak RSD , 2.24%, retention time ,3.63%, efficiency ,2.22% and asymmetry ,1.12%. Recovery The recovery of paroxetine from urine and serum were determined by spiking the drug-free samples, diluted in the ratio of 1:5 with the mobile phase and known amount of the drug at
three different concentrations within the calibration range. The spiked samples were processed and analyzed by six replicates with the developed procedure. The relative (analytical) recovery was calculated by comparing the concentration obtained from the drug supplemented sample with the actual added amounts; data obtained are recorded in Table IV. The results obtained show satisfactory recoveries of paroxetine in the range of 88– 97.5%.
Analysis of paroxetine in spiked and real blood samples Figure 2A and B shows the chromatograms of blank serum and urine, respectively. The analysis of real samples obtained from anonymous patients from a local hospital was performed (by three replicates) using small amount of serum or urine that was injected directly into the chromatographic system without any pretreatment other than filtration. If a sample contains more than the maximum quantifiable level (.5 mg/mL), it is diluted with water to reduce the concentration to reach the interval range. Results are shown in Table V. The obtained values coincide with the values obtained by the standard method of the Hospital. The chromatogram obtained during analysis of the serum Sample 1; containing 1.20 mg/mL paroxetine is shown in Figure 3.
Discussion The results demonstrated that the method was suitable for routine analysis of paroxetine in biological samples. The specificity studies have shown that the analyte can be detected without interferences in plasma and urine, despite the presence of endogenous compounds. The sensitivity and the calibration range are useful to detect the analyte at the levels that can be found in physiological fluids. The high linearity and recovery near 100% indicates that the values of concentration of paroxetine are near the true value. The low variability and high
Table IV. Determination of Paroxetine in Spiked Biological Samples at Three Different Concentrations (mg/mL) Using Electrochemical Detection Added concentration (mg/mL)
Found mean + SD (serum)
Found mean + SD (urine)
Recovery in serum (%)
Recovery in urine (%)
5 2.5 1
4.87 + 1.16 2.34 + 1.17 0.88 + 0.74
4.83 + 0.75 2.38 + 1.41 0.87 + 1.18
97.5 93.8 88
96.7 95.5 87
n ¼ 6.
Table III. Evaluation of the Robustness of the MLC Method (Average + SD; RSD, %) Parameters
Variation
Sensitivity (a.u.)
Retention time (min)
N (efficiency)
B/A (asymmetry)
Applied voltage (V) Flow rate (mL/min) Injection volume 1-Pentanol (%) SDS (M) pH
0.78 –0.82 0.9 –1.1 18 –22 5.5 –6.5 0.145 –0.155 2.8 –3.2
56.6 + 1.3 (2.24%) 56.4 + 0.6 (0.99%) 56.5 + 1.1 (1.87%) 56.5 + 0.4 (0.72%) 56.3 + 0.5 (0.81%) 56.4 + 0.6 (1.06%)
11.00 + 0.01 (0.09%) 11.0 + 0.2 (1.82%) 11.00 + 0.01 (0.09%) 11.0 + 0.4 (3.63%) 10.90 + 0.14 (1.30%) 11.00 + 0.01 (0.09%)
1790.7 + 1.5 (0.09%) 1792.7 + 2.1 (0.12%) 1789.7 + 2.1 (0.12%) 1800 + 40 (2.22%) 1790 + 7.0 (0.40%) 1791 + 1 (0.05%)
0.961 + 0.011 (1.12%) 0.974 + 0.004 (0.37%) 0.973 + 0.005 (0.46%) 0.977 + 0.004 (0.41%) 0.971 + 0.008 (0.79%) 0.9717 + 0.0016 (0.16%)
n ¼ 6.
Determination of Paroxetine in Blood 1221
environment-friendly, as it uses a smaller amount of toxic compounds, making it more attractive.
Table V. Determination of Paroxetine (mg/mL) in Serum and Urine of Patients Patient
Serum
Urine
1 2 3 4 5
1.20 + 0.04 4.54 + 0.08 Under LOD 8.15 + 0.06 15.04 + 0.09
0.84 + 0.03 2.15 + 0.09 Under LOD 6.52 + 0.04 12.34 + 0.07
Funding The work was supported by the project P1.1B2012-36, funded by the University Jaume I.
n ¼ 3.
Acknowledgments N.A. thanks University Grants Commission, M.H.R.D. (Government of India) for providing Junior Research Fellowship.
References
Figure 3. Chromatogram corresponding to real serum Sample 1, containing 1.20 mg/ mL paroxetine.
precision of the results obtained in different days are evident, which indicate the usefulness of the method. The developed method was found to be robust as the small deliberate changes in the method conditions did not have any significant effect on the chromatographic behavior of the analyte. The usefulness of the method has been assessed by the quantification of paroxetine in real samples from paroxetine takers, no interference has been observed and the levels of paroxetine have been determined with adequate precision.
Conclusion Analytical methods for the determination of paroxetine in serum and urine samples require extraction and preconcentration, which are two time and reagent consuming steps. Using the MLC-ECD method proposed here, the analyzed samples can be directly injected onto the chromatographic system and easily analyzed, the detection limits achieved to improve those obtained with conventional HPLC reported in the literature. The sensitivity of the method allows the monitorization of paroxetine in the biological samples and can be useful for physiological determinations. Validation was performed according to the ICH guidelines with satisfactory results in terms of linearity, selectivity, robustness, precision and accuracy. The lineal interval range was sufficient to detect the usual amount of paroxetine in patients’ serum and urine. The proposed methodology is cheap and requires low time of analysis (experimental procedure and chromatographic elution), making it suitable for routine analysis. Moreover, this method is relatively inexpensive and can be considered 1222 Agrawal et al.
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22. Bose, D., Durgbanshi, A., Martinavarro Dominguez, A., Capella Peiro´, M.E., Carda Broch, S., Esteve Romero, J., et al.; Rapid determination of acetaminophen in physiological fluids by liquid chromatography using SDS mobile phase and ED detection; Journal of Chromatographic Science, (2005); 43: 313–318. 23. Bose, D., Martinavarro Dominguez, A., Gil Agustı´ , M., Carda Broch, S., Durgbanshi, A., Capella Peiro´, M.E., et al.; Therapeutic monitoring of imipramine and desipramine by micellar liquid chromatography with direct injection and electrochemical detection; Biomedical Chromatography, (2005); 19(5): 343–349. 24. Bose, D., Durgbanshi, A., Martinavarro Dominguez, A., Capella Peiro´, M.E., Carda Broch, S., Esteve Romero, J., et al.; Amitriptyline and nortriptyline serum determination by micellar liquid chromatography; Journal of Pharmacological and Toxicological Methods, (2005); 52: 323–329. 25. Bose, D., Durgbanshi, A., Capella Peiro´, M.E., Gil Agustı´ , M., Esteve Romero, J., Carda Broch, S.; Micellar liquid chromatography determination of some biogenic amines with electrochemical detection; Journal of Pharmaceutical and Biomedical Analysis, (2004); 36: 357–363. 26. ICH validation guideline. http://www.ich.org/fileadmin/Public_Web_ Site/ICH_Products/Guidelines/Quality/Q2_R1/Step4/Q2_R1_Guideline. pdf (accessed December 23, 2013). 27. Esteve-Romero, J., Ochoa-Aranda, E., Bose, D., Rambla-Alegre, M., Peris-Vicente, J., Martinavarro-Bomı´ nguez, A.; Amoxifen monitoring studies in breast cancer patients by micellar liquid chromatography; Analytical Bioanalytical Chemistry, (2010); 397: 1557–1561. 28. Rambla-Alegre, M., Peris-Vicente, J., Marco-Peiro´, S., BeltranMartinavarro, B., Esteve-Romero, J.; Development of an analytical methodology to quantify melamine in milk using micellar liquid chromatography and validation according to EU Regulation 2002/ 654/EC; Talanta, (2010); 81: 894–900. 29. Rambla-Alegre, M., Peris-Vicente, J., Esteve-Romero, J., Carda-Broch, S.; Analysis of selected veterinary antibiotics in fish by micellar liquid chromatography with fluorescence detection and validation in accordance with regulation 2002/657/EC; Food Chemistry, (2010); 123: 1294–1302. 30. Berthod, A., Garcı´ a-A´lvarez-Coque, M.C. Micellar liquid chromatography, Cazes, J. (ed). Marcel-Dekker, New York, (2000).
Determination of Paroxetine in Blood 1223
Article
Determination of Paroxetine in Blood and Urine Using Micellar Liquid Chromatography with Electrochemical Detection Nitasha Agrawal1, Sergio Marco-Peiro´2, Josep Esteve-Romero2, Abhilasha Durgbanshi1, Devasish Bose1, Juan Peris-Vicente2* and Samuel Carda-Broch2 1
Department of Criminology and Forensic Sciences, Dr. H.S. Gour University, Sagar, India and 2A`rea de Quı´ mica Analı´ tica, Universitat Jaume I, Castello´ 12071, Spain
*Author to whom correspondence should be addressed. Email: [email protected] Received 30 April 2013; revised 23 September 2013
Paroxetine is a potent selective serotonin reuptake inhibitor used for the treatment of depression and related mood disorders. A micellar liquid chromatographic method was developed for the determination of paroxetine in serum and urine. Detection of paroxetine was carried out using a C18 column and a mobile phase of 0.15 M sodium dodecyl sulfate, 6% 1-pentanol at pH 3 (buffer salt 0.01 M NaH2PO4) running under isocratic mode at 1.0 mL/min and electrochemical detection at 0.8 V. The analyte was eluted without interferences in 0.9999; 0.5 –5 mg/mL range), accuracy (88–97.5%, recovery), repeatability (RSD < 0.54%), intermediate precision (RSD < 0.54%), limit of detection and quantification (0.001 and 0.005 mg/mL, respectively) and robustness (RSD < 3.63%). Developed method was successfully applied to real blood and urine samples as well as in spiked serum and urine samples. The developed method was specific, rapid, precise, reliable, accurate, inexpensive and then suitable for routine analysis of paroxetine in monitorized samples.
Introduction In the last few years, the prescription of antidepressants has increased many folds. The new drugs that enhance serotoninergic neurotransmission by potent and selective inhibition of serotonin are highest among the prescribed antidepressants, they are known as selective serotonin reuptake inhibitors (SSRI) (1). Paroxetine, (2)-trans-4-(4-fluorophenyl)-3-(3,4-methylenedioxy-phenoxymethyl) piperidine (Table I), is one of the SSRIs used widely as an antidepressant drug alone or in combination with other drugs (2). Paroxetine can be compared with the tricyclic antidepressants in their clinical efficacy; however, it is safer and has greater acceptance by the patients. Paroxetine is devoid of sedative effect and remarkably safe in overdose. It takes 5.2 h to reach the peak, with extended half-life (21 h) that allowed the introduction of formulations for once daily dosing. The above-mentioned qualities of paroxetine made the drug as the most widely prescribed antidepressant (3). It is used in the treatment of clinical depression, obsessive–compulsive disorder and panic disorder. In a survey performed by NDC Health (Atlanta, GA, USA) in 2011, paroxetine was one of the top 150 prescriptions for 2011 by number of US prescriptions dispensed (http://www.rxlist.com/script/main/ hp.asp).
Once administered, paroxetine is mainly oxidized to an unstable catechol followed by further methylation, and the major metabolite is conjugated rapidly to glucuronide and sulfate ethers in animals and humans. This major metabolite has not been found to inhibit serotonin reuptake significantly. Paroxetine and its metabolites in human plasma, serum or in tablets have been quantified by high-performance liquid chromatography (HPLC) (4). During the literature survey, it was also found that along with its benefits, paroxetine do possess some serious health risks. Patients with depression are at a high risk of suicide attempts (5). The European Medicines Agency’s Committee for Medicinal Products for Human Use (CHMP) published a letter for patients, prescribers and parents that paroxetine should not be prescribed to children. CHMP gave a warning to prescribers recommending close monitoring of adult patients at high risk of suicidal behavior and/or suicidal thoughts. The drug is often related with the withdrawal symptoms on stopping treatment abruptly and, therefore, it is recommended to gradually reduce the dose over several weeks or months if decision of withdrawal is made (6). A survey carried out by Food and Drug Administration in 2004 on children prescribed with paroxetine showed 2.7-fold raise in suicide behavior and ideation when compared with placebo. The trend for increased suicidality was observed in both trials for depression and for anxiety disorders (7). Several methods have been published for the determination of paroxetine in plasma, including reversed phase HPLC with UV (8, 9), fluorescence (with or without derivatization using dansylchloride) (10 –12), MS (13 –15), diode array detection (16, 17) or gas chromatography with MS detection (18 –20), and applied therapeutic drug level monitoring or bioequivalence studies. Only a few methods have been published for the simultaneous determination of paroxetine and its metabolites in plasma, including HPLC with UV (4, 8), and applied to pharmacokinetic, therapeutic drug level monitoring and toxicological screening or forensic cases. It was also noted that there were no papers published describing the use of micellar liquid chromatography (MLC) for the analysis of paroxetine using electrochemical detection (ECD). MLC is one of the most frequently used direct injection chromatographic technique which uses a surfactant at a concentration above the critical micellar concentration as mobile phase, presenting an alternative to conventional HPLC. The simultaneous elution of hydrophobic and hydrophilic analytes is possible without needing a gradient elution, and direct injection of physiological samples becomes feasible due to the solubility of proteins in the micelles, avoiding long and tedious extraction steps. MLC
# The Author [2014]. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
Table I. Structure and Physical Properties of Paroxetine Compound Paroxetine
Structure
pKa
Log Po/w
9.9
3.95
has been proved to be a useful technique in the determination of diverse group of compounds. In addition, the stable and reproducible behavior of micellar mobile phases allows accurate prediction of solute retention with a model that can also be used to optimize the separation of mixtures. MLC technique has been successfully used in the determination of large number of drugs in pharmaceutical preparations, biological fluids (serum and urine) that can be injected directly into the chromatographic system. Sodium dodecyl sulfate (SDS) is a widely used anionic surfactant in micellar media given its solubility in water, its low critic micellar concentration, low cost, and because it is easy to remove from the chromatographic system. The use of MLC expedites the experimental procedure, reducing the analysis time and the cost of the methodology (21–25). The purpose of the present work was to develop a rapid, simple and selective MLC procedure for the screening of paroxetine in urine, serum and physiological samples, using C18 column and ECD. The determination of paroxetine was simplified by direct injection of serum and urine samples in the MLC system, and the use of ECD reduces the protein band, thus increasing the selectivity. The proposed methodology was validated following the guidelines of the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) in serum in terms of specificity, linearity, accuracy, repeatability, intermediate precision, sensitivity (limits of detection and quantification, LOD and LOQ) and robustness (26). The suitability of the method to routine analysis of blood and urine samples of paroxetine takers was also considered.
Experimental Materials and reagents Standard paroxetine ( purity .99.99%) was kindly gifted from Laboratorio STADA, S.L. (Sant Just Desvern, Barcelona, Spain). SDS salt ( purity .99%) and potassium chloride ( purity .99%) was purchased from Merck (Darmstadt, Germany). Sodium dihydrogen phosphate ( purity .99%) and 37% HCl (from Panreac, Barcelona, Spain) and methanol, 1-propanol, 1-butanol and n-amyl alcohol (HPLC grade) were purchased from Scharlau (Barcelona, Spain). The serum was gifted by Lifeline pathology, Sagar, India and Hospital Provincial, Castellon, Spain. Urine was collected from healthy drug-free donors. Ultrapure water used throughout the study was obtained from distilled water by an Ultrapure Water device from Millipore S.A.S. (Molsheim, France). 1218 Agrawal et al.
Apparatus and instrumentation An Agilent Technologies Chromatograph (Model HP1100, Palo Alto, CA, USA), equipped with a quaternary pump, an autosampler and electrochemical (ECD, Model HP 1049A) detectors were used. Monitoring was performed at 800 mV with electrochemical detector with an Ag/AgCl as reference and glassy carbon as working electrode. A Kromasil C18 column (Scharlab, 5 mm particle size, 120 4.6 mm i.d.) was employed for the analytical separation. Flow rate and the injection volume were 1.0 mL/min and 20 mL, respectively. The dead time was determined as the mean value of the first significant deviation of the baseline in the chromatograms of the analytes. Signal was acquired by a PC connected to the chromatograph, through an HP Chemstation (Rev. B.03.01). An ultrasonic bath (model Ultrasons-H, Selecta, Abrera, Spain) was used to solve the standards and the pH of the solutions was measured with a Crison GLP 22 (Barcelona) potentiometer equipped with a combined Ag/AgCl/glass electrode. The analytical balance used was a Mettler-Toledo AX105 Delta-Range (Greifensee, Switzerland).
Sample preparation The drug sample was first weighed and then dissolved in methanol. The solutions were injected into the chromatograph without any pretreatment other than filtration, which was carried out directly into the autosampler vials through 0.45 mm nylon membranes. Both the serum and urine samples were first diluted in a ratio of 1:5 with the mobile phase before injection.
Mobile phase preparation Micellar mobile phases were prepared by weighing an exact amount ofSDS, sodium dihydrogen phosphate and KCl and dissolving them in water, adjusting the pH to 3 by adding drops of an HCl 6 M solution to reach the desired pH. A suitable volume of alcohol (i.e., 1-propanol, 1-butanol or 1-pentanol), depending on the percentage was added to the mobile phase as organic modifier. The usefulness of micellar mobile solvents and phases lies in the direct injection of physiological fluids as the monomers and micelles tend to bind proteins competitively; thereby releasing protein bound drugs which are free to partition into the stationary phase. Finally, the proteins, rather than precipitating into the column are solubilized and swept away harmlessly, eluting with or shortly after the solvent front. Due to the use of salt solutions as a mobile phase, special careful considerations are required to prevent the precipitation of SDS which would seriously damage the chromatographic system (27).
Results Method development and optimization Appropriate choice of stationary and mobile phase is very important for developing methods in micellar liquid chromatography. Selection of pH along with the appropriate oxidation potential is the first parameter to be studied when a micellar
procedure is developed using ECD. The amount of modifier in % plays a very significant role. Selection of oxidation potential Paroxetine is the only drug among SSRIs which is electrochemically active, which automatically filters it from other prescribed drugs of similar nature or structure. In order to establish the optimum oxidation potential, paroxetine was oxidized using 200 –900 mV, in steps of 100 up to 600 mV and then in steps of 50 mV. On the basis of the data obtained, 800 mV was selected for the analysis Figure 1. Selection of pH The pH variation of the mobile phase will affect the retention time of weak acids. Then pH must be fixed by the use of a buffer. The working pH range of the column is 1.5 –9.5, but the use of basic mobile phase causes the degradation of the C18 coating of the stationary phase, so that only acidic or neutral pH was considered (28). As pKa of paroxetine is 9.9 (Table I), at these pH, the analyte is under its neutral form. The analysis of paroxetine was carried out at pH 3, 5 and 7. The areas of the chromatographic peak of paroxetine were similar in the three cases, but in ECD, the micellar media get somewhat more oxidized at pH 7, resulting in a high background noise (baseline width: 14 mA for pH 7 and 6 mA for pH 3). Thus based on the value of pKa, structure and data obtained, it was decided to use pH 3 for further studies. Selection of organic modifier The partition coefficient of paroxetine (log Po/w ¼ 3.95; see Table I) suggests that the substance is highly hydrophobic. These kinds of compounds are not eluted in feasible times using pure aqueous micellar mobile phases and therefore need the addition of an organic modifier. Short-chain alcohols such as 1-propanol, 1-butanol and 1-pentanol could be appropriately
added to the mobile phase for achieving a higher elution strength that increases according to the length of their carbon chain (29). The concentration and the nature of the short-chain alcohol are critical in the separation. The use of higher concentrations of short-chain alcohol is not recommended in MLC. The alcohol tends to incorporate in the micelle structure (competing with SDS by the position of the micelle). This reduces the stability of the micelle and favors its destruction. Therefore, the number of micelles is lower than the expected and not reproducible, disturbing the analysis. The affinity of the alcohol with the micelle increases with the hydrophobicity of the alcohol. Therefore, the maximum tolerable concentration is increased when the carbon chain is shorter. Maximum tolerated concentrations for each short-chain alcohol are the values indicated in Table II (30). To select the organic modifier, a serum sample free of paroxetine and a blank serum sample spiked with 2.5 mg/mL were analyzed (by six replicates). Three mobile phases with maximum concentration of SDS (0.15 M) and a high concentration of alcohol (12% 1-propanol, 7% 1-butanol and 6% 1-pentanol) were tested. The retention time, retention factor (k0 ), efficiency (N) and asymmetry (B/A) of the chromatographic peak corresponding to paroxetine were determined for each tested mobile phase, and the results are shown in Table II. Effectively, elution time was lower and efficiency higher using short-chain alcohols at increasing molecular weight. Finally, the optimum mobile phase selected was based on the minimum analysis time, high resolution and adequate peak shape. The selected mobile phase was 0.15 M SDS– 6% 1-pentanol at pH 3. Under these conditions, the chromatogram sample serum free of paroxetine shows a wide front (to 4 min), but no other peak was observed at higher elution times (Fig. 2A). In the chromatogram from spiked serum sample, the chromatographic peak of paroxetine appears at 11.00 + 0.04 min without interferences and shows an adequate shape (Fig. 2C). The retention time was low but enough far from the dead time (4 min), which is useful for routine analysis.
Validation The developed method was validated for specificity, accuracy, linearity, LOD, LOQ, repeatability, intermediate precision and robustness. The method was developed in serum and urine. As results are similar for both cases, the values for serum are shown. Specificity The specificity is the ability to assess unequivocally the analyte in the presence of other drugs which can be prescribed together or other compounds of the matrices, especially proteins. In the
Table II. Chromatographic Parameter of the Peak Corresponding to Paroxetine at Different Organic Modifier Added to a 0.15 M SDS Aqueous Mobile Phase Buffered at pH 3
Figure 1. Chromatograms obtained by analyzing a standard solution 5 mg/mL of paroxetine by MLC-ECD at several oxidation potential.
Organic modifier (amount)
Retention time (min)
k0 (retention factor)
N (efficiency)
B/A (asymmetry)
1-Propanol (12%) 1-Butanol (7%) 1-Pentanol (6%)
31.6 + 0.3 22.46 + 0.05 11.00 + 0.04
32.26 22.64 10.57
1289 + 3 1546.5 + 0.9 1790.8 + 0.5
0.916 + 0.007 1.124 + 0.008 0.974 + 0.005
n ¼ 6.
Determination of Paroxetine in Blood 1219
Figure 2. Obtained chromatogram using 0.15 M SDS, 6% 1-pentanol at pH 3, 1.0 mL/min and ECD 0.8 V for injection of (A) blank serum, (B) blank urine, (C) serum spiked with 2.5 mg/mL paroxetine and (D) urine spiked with 2.5 mg/mL.
case of chromatographic analysis, it should be assessed that there is not compounds in the matrices with the same retention time. As has been previously indicated (Selection of oxidation potential section), SSRIs (except paroxetine) are not electrochemically active and then could not interfere with the quantification of the analyte. On the other hand, micellar mobile phases provoke the solubilization of proteins and their elution at the dead time (,4 min) (27). Paroxetine-free serum and urine samples were selected as controls and processed directly in the chromatographic system. They were then analyzed to determine the presence in serum (Fig. 2A) and urine (Fig. 2B) of endogenous components which can overlap with the analyte. Then they were compared with spiked samples with 2.5 mg/mL to evaluate the specificity of the 1220 Agrawal et al.
method. No interference from endogenous compounds was found in serum (Fig. 2C) neither in urine (Fig. 2D). Linearity and limit of detection For linearity study, paroxetine was injected at different concentrations after dilution of the stock solution with matrix samples free of paroxetine. Under the selected chromatographic conditions, the linear range of the signal response for the drug was studied in the concentration ranging from 0.05 to 5 mg/mL at nine different concentration levels, analysis of each calibration level was replicated six times. The slope, intercept and regression coefficient (r 2) were determined by plotting the area of the chromatographic peak of the paroxetine versus the concentration using the least-square linear regression analysis method. To study the variability of the calibration parameters, five curves
were constructed by 5 days over a 2-month period using different sets of standard, and the average values were shown. The adjusted equations and regression coefficients for paroxetine in serum were: A ða.u.Þ ¼ ð55:977 + 0:008Þ ½ paroxetine ðmg/mL) þ ð0:4 + 0:5Þr 2 . 0:9999 The LOD and LOQ for the drug were determined with the 3 s criterion and 10 s criterion, respectively (as indicated in ICH guidelines), using a series of 10 solutions containing low concentrations (0.05 –5 mg/mL) of the drug. The results were based not only on the standard deviation of the response but also on the slope of a specific calibration curve containing the analyte. Under these conditions, the LOD and LOQ were 0.001 and 0.05 mg/mL, respectively. Repeatability and intermediate precision Repeatability and intermediate precision of the suggested methodology were determined using blank matrix samples spiked at three concentration levels: 1, 2.5 and 5 mg/mL. Repeatability was determined by analyzing six replicated of each concentration in the same day and calculating RSD of the areas of the chromatographic peak corresponding to paroxetine (intraday precision), whereas intermediate precision correspond to the average of five measurements of the intraday values over a 3-month period (interday precision). The results for 1, 2.5 and 5 mg/mL were: repeatability 0.13, 0.54 and 0.35%, respectively, and intermediate precision 0.17, 0.16 and 0.54%, respectively. Robustness The robustness was evaluated by analyzing a blank serum spiked with 1 mg/mL of paroxetine standard (n ¼ 6) by making slight deliberate changes to the following parameters: SDS concentration, 1-pentanol amount, pH, applied voltage, flow rate and injection volume. The variation of sensitivity (area of chromatographic peak), retention time, efficiency and asymmetry was measured as RSD, and the results are shown in Table III. The variations in all the studied chromatographic parameters were: area of chromatographic peak RSD , 2.24%, retention time ,3.63%, efficiency ,2.22% and asymmetry ,1.12%. Recovery The recovery of paroxetine from urine and serum were determined by spiking the drug-free samples, diluted in the ratio of 1:5 with the mobile phase and known amount of the drug at
three different concentrations within the calibration range. The spiked samples were processed and analyzed by six replicates with the developed procedure. The relative (analytical) recovery was calculated by comparing the concentration obtained from the drug supplemented sample with the actual added amounts; data obtained are recorded in Table IV. The results obtained show satisfactory recoveries of paroxetine in the range of 88– 97.5%.
Analysis of paroxetine in spiked and real blood samples Figure 2A and B shows the chromatograms of blank serum and urine, respectively. The analysis of real samples obtained from anonymous patients from a local hospital was performed (by three replicates) using small amount of serum or urine that was injected directly into the chromatographic system without any pretreatment other than filtration. If a sample contains more than the maximum quantifiable level (.5 mg/mL), it is diluted with water to reduce the concentration to reach the interval range. Results are shown in Table V. The obtained values coincide with the values obtained by the standard method of the Hospital. The chromatogram obtained during analysis of the serum Sample 1; containing 1.20 mg/mL paroxetine is shown in Figure 3.
Discussion The results demonstrated that the method was suitable for routine analysis of paroxetine in biological samples. The specificity studies have shown that the analyte can be detected without interferences in plasma and urine, despite the presence of endogenous compounds. The sensitivity and the calibration range are useful to detect the analyte at the levels that can be found in physiological fluids. The high linearity and recovery near 100% indicates that the values of concentration of paroxetine are near the true value. The low variability and high
Table IV. Determination of Paroxetine in Spiked Biological Samples at Three Different Concentrations (mg/mL) Using Electrochemical Detection Added concentration (mg/mL)
Found mean + SD (serum)
Found mean + SD (urine)
Recovery in serum (%)
Recovery in urine (%)
5 2.5 1
4.87 + 1.16 2.34 + 1.17 0.88 + 0.74
4.83 + 0.75 2.38 + 1.41 0.87 + 1.18
97.5 93.8 88
96.7 95.5 87
n ¼ 6.
Table III. Evaluation of the Robustness of the MLC Method (Average + SD; RSD, %) Parameters
Variation
Sensitivity (a.u.)
Retention time (min)
N (efficiency)
B/A (asymmetry)
Applied voltage (V) Flow rate (mL/min) Injection volume 1-Pentanol (%) SDS (M) pH
0.78 –0.82 0.9 –1.1 18 –22 5.5 –6.5 0.145 –0.155 2.8 –3.2
56.6 + 1.3 (2.24%) 56.4 + 0.6 (0.99%) 56.5 + 1.1 (1.87%) 56.5 + 0.4 (0.72%) 56.3 + 0.5 (0.81%) 56.4 + 0.6 (1.06%)
11.00 + 0.01 (0.09%) 11.0 + 0.2 (1.82%) 11.00 + 0.01 (0.09%) 11.0 + 0.4 (3.63%) 10.90 + 0.14 (1.30%) 11.00 + 0.01 (0.09%)
1790.7 + 1.5 (0.09%) 1792.7 + 2.1 (0.12%) 1789.7 + 2.1 (0.12%) 1800 + 40 (2.22%) 1790 + 7.0 (0.40%) 1791 + 1 (0.05%)
0.961 + 0.011 (1.12%) 0.974 + 0.004 (0.37%) 0.973 + 0.005 (0.46%) 0.977 + 0.004 (0.41%) 0.971 + 0.008 (0.79%) 0.9717 + 0.0016 (0.16%)
n ¼ 6.
Determination of Paroxetine in Blood 1221
environment-friendly, as it uses a smaller amount of toxic compounds, making it more attractive.
Table V. Determination of Paroxetine (mg/mL) in Serum and Urine of Patients Patient
Serum
Urine
1 2 3 4 5
1.20 + 0.04 4.54 + 0.08 Under LOD 8.15 + 0.06 15.04 + 0.09
0.84 + 0.03 2.15 + 0.09 Under LOD 6.52 + 0.04 12.34 + 0.07
Funding The work was supported by the project P1.1B2012-36, funded by the University Jaume I.
n ¼ 3.
Acknowledgments N.A. thanks University Grants Commission, M.H.R.D. (Government of India) for providing Junior Research Fellowship.
References
Figure 3. Chromatogram corresponding to real serum Sample 1, containing 1.20 mg/ mL paroxetine.
precision of the results obtained in different days are evident, which indicate the usefulness of the method. The developed method was found to be robust as the small deliberate changes in the method conditions did not have any significant effect on the chromatographic behavior of the analyte. The usefulness of the method has been assessed by the quantification of paroxetine in real samples from paroxetine takers, no interference has been observed and the levels of paroxetine have been determined with adequate precision.
Conclusion Analytical methods for the determination of paroxetine in serum and urine samples require extraction and preconcentration, which are two time and reagent consuming steps. Using the MLC-ECD method proposed here, the analyzed samples can be directly injected onto the chromatographic system and easily analyzed, the detection limits achieved to improve those obtained with conventional HPLC reported in the literature. The sensitivity of the method allows the monitorization of paroxetine in the biological samples and can be useful for physiological determinations. Validation was performed according to the ICH guidelines with satisfactory results in terms of linearity, selectivity, robustness, precision and accuracy. The lineal interval range was sufficient to detect the usual amount of paroxetine in patients’ serum and urine. The proposed methodology is cheap and requires low time of analysis (experimental procedure and chromatographic elution), making it suitable for routine analysis. Moreover, this method is relatively inexpensive and can be considered 1222 Agrawal et al.
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