J S S
ISSN 1615-9306 · JSSCCJ 38 (9) 1441–1624 (2015) · Vol. 38 · No. 9 · May 2015 · D 10609
JOURNAL OF
SEPARATION SCIENCE
Methods Chromatography · Electroseparation Applications Biomedicine · Foods · Environment
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Sai Sandeep Mannemala1,2 Janaki Sankarachari Krishnan Nagarajan1 1 Department
of Pharmaceutical Analysis, JSS University, Udhagamandalam, Tamil Nadu, India 2 Department of Pharmacy, Annamalai University, Annamalai Nagar, Tamil Nadu, India Received December 25, 2014 Revised February 3, 2015 Accepted February 5, 2015
Research Article
Development and validation of a generic liquid chromatographic method for the simultaneous determination of five commonly used antimalarial drugs: Application to pharmaceutical formulations and human plasma A simple, sensitive, and rapid liquid chromatographic method was developed and validated using diode array detection for the determination of five commonly used antimalarial drugs in pharmaceutical formulations and in human plasma. Chromatographic separation of antimalarial drugs and internal standard (ibuprofen) was achieved on a C18 column with a mobile phase composed of 10 mM dipotassium orthophosphate at pH 3.0, methanol, and acetonitrile in a ratio of 20:38:42 v/v, at a flow rate of 1 mL/min. The analytes were monitored at 220 nm and separated in ˂10 min. The method was validated for linearity, accuracy, precision, limit of quantification, and robustness. Both intra- and interday precisions (in terms of %RSD) were lower than 3% and accuracy ranged from 98.1 to 104.5%. Extraction recoveries were ࣙ96% in plasma. The limits of quantitation for artemether, lumefantrine, pyrimethamine, sulfadoxine, and mefloquine were 0.3, 0.03, 0.06, 0.15, and 0.15 g/mL in human plasma. Stability under various conditions was also investigated. The method was successfully applied for quantification of antimalarial drugs in marketed formulations and in spiked human plasma. The method can be employed for routine QC purposes and in pharmacokinetic investigations. Keywords: Antimalarial drugs / Bioanalytical methods / Fractional factorial design / High-performance liquid chromatography / Human plasma DOI 10.1002/jssc.201401465
Additional supporting information may be found in the online version of this article at the publisher’s web-site
1 Introduction Malaria is one of the most common infectious diseases and a great public health issue worldwide, predominantly in Africa and South Asia. Although, malaria is preventable and treatable, it continues to cause more than 600 000 deaths per year [1]. The emergence of drug-resistant strains of Plasmodium became foremost complication in control of the disease which threatens the effectiveness of treatment. To combat the infection, combination therapies are highly advocated. The current first-line drug therapy for chloroquine-resistant malaria is sulfadoxine (SFD) and pyrimethamine (PYT) Correspondence: Sai Sandeep Mannemala, Department of Pharmacy, Annamalai University, Annamalai Nagar, Chidambaram, Tamil Nadu 608001, India E-mail: [email protected]
Abbreviations: ART, artemether; IBU, ibuprofen; IS, internal standard; LFT, lumefantrine; MFL, mefloquine; PYT, pyrimethamine; SFD, sulfadoxine
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with mefloquine (MFL) or parallel courses with quinine and doxycycline as second-line of medication [2]. In cases of multidrug resistance, the fixed combination of artemether (ART) and lumefantrine (LFT) is recommended with MFL [3]. One major concern in the treatment of malaria is the production of substandard and counterfeited drugs which pose an immediate threat to public health and undermine malaria control efforts. The counterfeited drugs result in declined therapeutic efficacy, which consecutively risk the drugs to reach the effective therapeutic concentration levels required for elimination of the parasites. About 15% of all antimalarial drugs in circulation worldwide are believed to be counterfeited, with the figures rising to as high as 50% in some parts of Africa and Asia [4]. So, it is indeed important to control the quality of antimalarial drugs. The combination therapy also demands sensitive and selective analytical methods for the simultaneous quantification of these drugs in biological matrices from a pharmaceutical perspective means of understanding pharmacokinetics and therapeutic drug monitoring.
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This requires validated methods that are sensitive, accurate, and precise. ART (Supporting Information Fig. S1a), (3R,5aS,6R,8aS,9R,10S,12R,12aR)-10-methoxy-3,6,9trimethyldecahydro-12H-3,12-epoxy[1,2]dioxepino[4,3-i] 2-benzopyran, is a methyl ethyl derivative of artemisinin, which is highly effective against the blood schizonts of both malarial parasites Plasmodium falciparum and Plasmodium vivax. LFT (Supporting Information Fig. S1b), 2(dibutylamino)-1-[(9Z)-2,7-dichloro-9-(4-chlorobenzylidene)9H-fluoren-4-yl]ethanol, is used to treat acute uncomplicated malaria in combination with ART to improve efficacy in infections acquired in chloroquine-resistant strains and its biological activity is related to class-2 arylamino alcohols. PYT (Supporting Information Fig. S1c), 5-(4-chlorophenyl)6-ethyl-2,4-pyrimidinediamine, is a derivative of diaminopyrimidine, which is used in combination with SFD in the treatment of malaria. SFD (Supporting Information Fig. S1d), 4-amino-N-(5,6-dimethoxy-4-pyrimidinyl)benzene sulfonamide, is a sulfonamide derivative used in the treatment of malaria. MFL hydrochloride (Fig. 1e), (R,S)-2,8[bis(trifluoromethyl)quinolin-4-yl]-(2-piperidyl) methanol, is 2-aryl substituted analogue of quinine, which is used in second-line treatment of chloroquine-resistant P. falciparum malaria. Ibuprofen (IBU; Supporting Information Fig. S1f), (RS)-2-[4-(2-methylpropyl)phenyl]propanoic acid, is a nonsteroidal anti-inflammatory drug and derivative of propionic acid used for relieving pain and fever and reducing inflammation. Several methods have been reported for determination of ART and LFT in pharmaceutical formulations by HPLC coupled with UV detection [5–10] or MS detection [11–13]. Additionally, there are many methods reported for the determination of PYT and SFD in pharmaceutical formulations and biological matrices by UV spectrophotometry [14, 15], by HPLC coupled with UV [16–20] or MS detection [21, 22]. Lamalle et al. [23] developed a generic micellar electrokinectic chromatographic method for the separation of 15 antimalarial drugs. The reported method does not include the determination of LFT and electrophoretic methods cannot be employed for routine QC due to economic considerations. Hodel et al. [11] reported an LC–MS/MS method for the determination of 14 antimalarial drugs and their metabolites. The method employs a complex gradient elution which involves longer equilibration time (ࣈ21 min) and chromatographic run time (16 min). Debrus et al. [13] proposed an innovative HPLC method for screening 19 antimalarial drugs using design of experiments approach. Although, the method uses novel Quality by Design methodology, it employs a complex gradient elution and requires higher run time for the analysis. Moreover, none of the above proposed methods have been employed for determination of antimalarials in pharmaceutical formulations or biological matrices. Together, these studies highlight the demand of a generic HPLC–PDA method which is simple, short, precise, robust, and accurate as well as cost-effective for the analysis of selected antimalarial drugs. Although one patient would not be prescribed all antimalarial C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
drugs under study at once, the proposed method has the advantages of providing suitability for routine clinical investigations, bioequivalence studies, and drug–drug interaction studies which demands detection of more than one analyte in biological matrices. The present study describes the development of a simple, economic, and sensitive HPLC–PDA method for simultaneous determination of ART, LFT, PYT, SFD, and MFL in pharmaceutical preparations and human plasma. Sample pretreatment involved a simple protein precipitation technique. The method was validated and was applied for QC of the formulations and determination in human plasma. To our knowledge, this is the first report of the HPLC–PDA method for the simultaneous determination of the selected antimalarials in formulation and human plasma with UV detection in shorter runtime.
2 Materials and methods 2.1 Chemicals and reagents Pure standards of ART (99.1%) and LFT (99.3%) were generously donated by Medreich Limited (Karnataka, India), PYT (98.6%) and SFD (99.5%) were provided by Nosch labs (Andhra Pradesh, India), MFL (98.9%) and IBU (99.4%; internal standard (IS)) were a generous gift from Micro Labs (Bangalore, India). Acetonitrile of HPLC grade was purchased from Sigma–Aldrich (Bangalore, India), dipotassium orthophosphate and phosphoric acid of AR grade were purchased from SD fine chemicals (Mumbai, India) and Milli-Q water was prepared from Milli-Q Academic, Millipore (Bangalore, India). The tablets Arte plus (Zydus Cadila Healthcare, India), Coartrin (Medreich Saimirra, India), and Arlufe (Neiss Labs, India) containing ART and LFT; Amalar Forte (Micro Labs, India), Croyodoxin FM (Glaxo Smithkline pharmaceuticals, India), and Malocide (Torrent Labs, India) containing PYT and SFD; Confal (Lupin laboratories, India), Mefliam (Cipla, India), and MFL (Emcure Pharmaceuticals, India) containing MEF were purchased from local pharmacy.
2.2 Plasma sampling Drug-free human blood samples were collected from healthy volunteers in tubes containing sodium citrate solution. The samples were centrifuged for 10 min at 1500 rpm for separation of plasma from blood. Subsequently, the samples were transferred to polypropylene tubes and stored at –20⬚C until further use.
2.3 Instrumentation and chromatographic conditions Chromatographic measurements were performed using a Shimadzu HPLC (Shimadzu, Tokyo, Japan) model equipped with Shimadzu LC-10 AT VP solvent delivery module www.jss-journal.com
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Figure 1. Representative chromatograms corresponding to (A) a synthetic mixture containing PYT, SFD, ART, LFT, MFL, and IBU (IS), under optimal conditions; (B) processed blank human plasma; (C) drugs PYT, SFD, ART, LFT, and MFL spiked in human plasma with IBU (IS).
with SPD M-10AVP PDA detector. Data acquisition was performed using Shimadzu Class-VP software. Chromatographic analysis was carried out using Phenomenex Gemini C18 Column (150 × 4.6 mm, 5m; Phenomenex , USA). The mobile phase was a mixture of dipotassium orthophosphate R
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buffer/acetonitrile/methanol in ratio of 20:38:42 v/v adjusted to a pH of 3.0 using phosphoric acid. The mobile phase was filtered through 0.22 m membrane filter (Gelman, India) and degassed ultrasonically for 15 min before use. The flow rate was set at 1 mL/min. A sample volume of 20 L was www.jss-journal.com
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injected for the chromatographic analysis. Based on the obtained isobestic point, the detector was set at 220 nm for the determination of all the analytes.
2.4 Analysis of tablets
ples (limit of quantification (LOQ), low QC, medium QC, and high QC) were prepared in blank human plasma at concentrations of 0.3, 1, 2.5, and 5 g/mL for ART; 0.03, 0.1, 0.25, and 0.5 g/mL for LFT; 0.06, 0.2, 0.5, and 1 g/mL for PYT; 0.15, 0.5, 1.5, and 2.5 g/mL for SFD and MFL. All samples were stored at –70⬚C until further use.
2.4.1 Preparation of stock and standard solutions
2.5.2 Plasma sample preparation
The standard stock solutions of ART, LFT, PYT, SFD, MFL, and IBU at 1000 g/mL were prepared separately by dissolving appropriate amounts of each compound in acetonitrile and methanol mixture, except for LFT in which 2 mL of chloroform is added to ensure its complete solubility. Working standard solutions were prepared by diluting the stock solutions with mobile phase to obtain a concentration range of 10, 15, 20, 25, and 30 g/mL for ART; 20, 40, 60, 80, and 100 g/mL for LFT, PYT, and MFL; 2, 4, 6, 8, and 10 g/mL for SFD.
During the day of analysis, the samples were removed from the deep freezer and were allowed to thaw. 250 L of aliquots were transferred to polypropylene tubes, spiked with 50 L of IBU (5 g/mL) as IS and mixed for 30 s. To the mixture, 300 L of acetonitrile was added and was vortex mixed for 3 min. After centrifugation at 1000 rpm for 10 min, the supernatant was transferred to polypropylene tubes. The collected supernatant was filtered using 0.22 m membrane filter and the filtrate was evaporated to dryness under the stream of nitrogen gas. The residue was reconstituted with 300 L of the mobile phase and a 20-L aliquot was injected into the HPLC system.
2.4.2 Preparation of tablet placebo The analytical placebo solutions were prepared by dissolving the stated excipients in selected tablet formulations in ethanol and were used for validation. 2.4.3 Preparation of tablet sample solutions For assay, 20 tablets containing (ART + LFT), (PYT + SFD), and MFL were weighed and powdered individually. An amount of tablet powder equivalent to 50 mg was weighed and transferred to a 50 mL volumetric flask followed by addition of mobile phase and IS. The mixture was subjected to sonication for 20 min and the solution was made up to the mark with the mobile phase. The solutions were centrifuged at 4000 rpm for 10 min. The clear supernatant was collected and filtered through a 0.22 m membrane filter. An aliquot of 20 L of this solution was used for HPLC analysis.
2.5 Analysis of plasma samples 2.5.1 Preparation of working standard solutions and QC samples The stock solutions of the antimalarial drugs and IS were prepared according to the procedure described in Section 2.4.1. The stock solutions were further diluted five- to 20-fold with methanol and acetonitrile mixture to prepare a series of working standard solutions. Calibration standards were prepared by spiking 20 L of prepared working standard solutions to 230 L human plasma to produce 0.3, 0.6, 0.9, 1.8, 2.4, 3.6, 4.8, and 6 g/mL for ART; 0.03, 0.06, 0.09, 0.18, 0.24, 0.36, 0.48, and 0.6 g/mL for LFT; 0.06, 0.12, 0.18, 0.36, 0.48, 0.72, 0.96, and 1.2 g/mL for PYT; 0.15, 0.3, 0.45, 0.9, 1.2, 1.8, 2.4, and 3 g/mL for SFD and MFL. Four levels of QC sam C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
2.6 Statistical tools Experimental design and Pareto chart analysis was performed by using Statgraphics Centurion XVI, trial version 16.2 (StatPoint Technologies, Warrenton, VA, USA).
2.7 Method validation The developed HPLC–PDA method for the determination of antimalarials in tablets was validated in compliance ICH guidelines [24], while the validation of method for the determination of antimalarials in human plasma was performed by following the USFDA-CDER guidelines [25]. The tested parameters were selectivity, accuracy, precision, linearity, recovery, LOD, LOQ, and stability. 2.7.1 Selectivity and specificity The specificity of the method for determination of antimalarials in tablets was tested by comparing the chromatograms obtained by analyzing the prepared placebo sample with that of the selected antimalarial drugs. Furthermore, the selectivity of the method was evaluated by comparing the peak purity plots obtained for analytes in both sample and standard solutions [24]. The selectivity of the method for determination of antimalarials in human plasma was assessed by analyzing the presence of potential chromatographic interferences of plasma endogenous compounds at the retention times of PYT, SFD, MFL, ART, LFT, and IS. For this, the blank plasma samples obtained from six different subjects was processed and the chromatograms were compared with spiked plasma samples [26]. www.jss-journal.com
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2.7.2 Linearity The linearity of the developed analytical method for tablet matrix was established by analyzing five concentrations each over a range of 5–30 g/mL for ART, 2–10 g/mL for SFD, 10–50 g/mL for MFL, and 20–100 g/mL for LFT and PYT. Six replicates per concentration were injected and chromatograms were recorded. Calibration curves were plotted by using recorded peak area ratios of the drug versus nominal concentration of drug. The acceptance criterion for each back-calculated standard concentration was 100 ± 2% with %RSD lower than 2% [24]. The linearity of the developed bioanalytical method was tested by analyzing eight nonzero calibration standards of 0.3–6 g/mL for ART, 0.03–0.6 g/mL for LFT, 0.06– 1.2 g/mL for PYT, 0.15–3 g/mL for SFD and MFL. Six replicates per concentration were injected and chromatograms were recorded. The acceptance criterion for each backcalculated standard concentration was 100 ± 10% (%RSD ࣘ 10%) except at the LOQ concentration, where the %RSD should not exceed 15% [25]. 2.7.3 Accuracy and precision The accuracy and precision of the analytical method was evaluated in five replicates at three concentration levels 80, 100, and 120% by spiking the known amount of working standard solutions to the tablet placebo. The criteria for the acceptability of the accuracy data are ±2% deviation from the nominal value and for precision within ±2 %RSD [24]. All the samples were analyzed in five replicates on three consecutive days. The accuracy and precision of the proposed bioanalytical method was evaluated at four concentration levels covering the entire linearity range including the LOQ concentration. All the samples were analyzed in five replicates on three consecutive days. The criteria for the acceptability of the accuracy data are ±15% deviation from the nominal value and for precision within ±15 %RSD [25]. 2.7.4 Recovery The absolute recoveries of antimalarial drugs from human plasma were assessed at four concentration levels from 0.3 to 0.5 g/mL for ART, 0.03 to 0.5 g/mL for LFT, 0.06 to 1 g/mL for PYT, 0.15 to 2.5 g/mL for SFD and MFL. The recoveries of the analytes were calculated by comparing the analyte peak area ratios from processed plasma samples with that the corresponding ratios obtained from the aqueous samples at same concentrations [27]. 2.7.5 Stability 2.7.5.1 Tablet solution stability It is necessary to evaluate the stability of both the standard and tablet sample solutions during the entire analytical process. For this, both standard and sample solutions were analyzed C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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over a period of 72 h at 25⬚C. The solutions were considered to be stable if the assay results were within acceptable accuracy (100 ± 2%) and precision (± 2 %RSD). 2.7.5.2 Freeze-thaw stability The stability of antimalarial drugs in human plasma was investigated for three freeze-thaw cycles, at 25⬚C for 3 h and at –70⬚C for four weeks by analyzing prepared QC samples in five replicates each to simulate sample handling and storage time in the freezer before the analysis [28]. The stability was assessed by comparing the mean concentrations of the stability sample with theoretical concentrations. The samples were considered stable if the assay results were within acceptable accuracy (100 ± 10%) [29]. 2.7.6 Robustness testing In this study, the robustness of the method was tested by employing fractional factorial design (24–1 ) [30]. The robustness of the method was tested using the following variables such as concentration of acetonitrile (range: 36–40% v/v), pH (range: 2.9–3.1), strength of buffer (range: 8–12 mM), and flow rate (range: 0.9–1.1 mL/min). Variables that were identified to be critical for the quality of the separation for current analysis such as retention factor of first peak, resolution between critical peak pair, and total analysis time were selected as responses.
3 Results and discussion 3.1 Optimization of chromatographic conditions In this study, a generic approach for the simultaneous determination of five commonly used antimalarial drugs by HPLC was developed. Based on the chemical nature of the analytes, preliminary experiments were conducted by using a C18 column and mobile phase comprising acetonitrile and water in 50:50 ratio. The pH was adjusted to 4 using phosphoric acid. At these conditions, the resolution between PYT and SFD was not satisfactory. Additionally, an increase in the buffer content also showed a remarkable increase in retention of LFT. The optimization of mobile phase composition was based on obtaining the maximum resolution (ࣙ2) and minimum run time ( 0.05) was found between the variances.
3.2 Method validation 3.2.3 Accuracy and precision 3.2.1 Selectivity and specificity The selectivity of the method was assessed by applying the proposed chromatographic procedure to tablets placebo and six different lots of plasma. No interfering peaks were found at the retention times of the selected analytes, indicating no interference from tablet excipients. Additionally, the peak purity angles were within the threshold limits indicating that no peaks were co-eluted with the analytes. Furthermore, from Fig. 1B and C, it can be observed that no endogenous interferences were found in retention times of antimalarials extracted from plasma. 3.2.2 Linearity The obtained regression equations for calibration curves were as follows: y = 0.0017x–0.0013 for ART, y = 0.0034x + 0.001 for LFT, y = 0.0044x + 0.0008 for PYT, y = 0.0016x + 0.0011 for SFD, and y = 0.0081x–0.0462 for MFL. The obtained correlation coefficients for all calibration curves were consistently greater than 0.9990 ± 0.0007 demonstrating good linearity over the entire concentration range. The linearity of the developed bioanalytical method was evaluated using eight nonzero concentrations in six replicates. For determination of antimalarials in human plasma, C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
The accuracy of the developed method was confirmed by computing the percentage recovery of known added amounts of antimalarials at three levels in to a blank matrix (tablet placebo or blank plasma. From Table 2, it can be seen that the recoveries of antimalarials at each level were found to be 98.08– 101.92% from tablet placebo, which lies within the acceptable criteria of the bias ±2%. The intraday and interday precision for developed analytical method was evaluated by analyzing five replicates of PYT, SFD, ART, LFT, and MFL at three different concentration levels and the results are expressed as %RSD (Table 2). The low value of %RSD showed that the method is precise within the acceptance limit of ±2%. From Table 1, it can be seen that both the intraday and interday precision data for the five replicate determinations were within the acceptance range of ±15 %RSD (>3.2%) and accuracy data were within the acceptance range of ±15% (98.1–104.5%) deviation from nominal value. 3.2.4 Recovery From Supporting Information Table S1, it can be seen that the mean recoveries of PYT, SFD, ART, LFT, and MFL were ranged from 96.67 to 102.19, 97.33 to 102.67, 96.69 to 100.46, 96.06 to 103.33, and 98.67 to 101.4%, respectively. The www.jss-journal.com
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Analyte PYT
SFD
ART
LFT
MFL
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a shift in retention times, but this did not affect the peak symmetry and peak areas of the analytes.
Accuracy (%)
Precision (%RSD)
3.2.7 Stability
Concentration (g/mL)
Intraday
Interday
Intraday
Interday
60 80 100 6 8 10 20 25 30 60 80 100 60 80 100
99.25 98.08 98.25 101.39 100.2 99.34 99.41 99.25 98.81 100.86 101.50 98.75 101.07 99.40 98.31
101.6 99.15 98.76 101.49 98.71 99.58 98.17 100.80 99.43 99.18 100.47 99.67 99.83 100.73 101.92
1.41 1.13 0.59 1.24 1.76 0.44 1.19 0.72 1.43 1.27 1.08 0.67 1.01 0.64 0.97
1.67 1.54 1.22 1.03 1.69 0.98 1.54 1.06 1.18 0.69 1.82 1.73 0.24 1.45 1.58
3.2.7.1 Tablet solution stability The tablet and standard solutions were analyzed over a period of 72 h and the results from the stability studies indicate that the peak areas of the analytes remain unaffected by showing %RSD ˂ 1.43 with an accuracy of 100 ± 1.72. No significant degradation was found within the period of evaluation, indicating the solutions were stable. 3.2.7.2 Freeze-thaw stability Three freeze-thaw cycles, short-term stability at 25⬚C for 3 h, and long-term stability at –70⬚C for four weeks showed no significant effect on the stability of QC samples of antimalarials. The results from Supporting Information Table S3 suggest that the antimalarials were stable in plasma when stored in frozen state for the stipulated period of time.
4 Application to commercial tablets recovery of IBU (IS) was calculated and a mean value of 98.4% was obtained. Furthermore, the %RSD values for recoveries of PYT, SFD, ART, LFT, and MFL were very low (ࣘ3.01%), suggesting that the proposed procedure was consistent and efficient in the proposed concentration range. 3.2.5 LOD and quantification The LOD and LOQ were established for all the analytes by measuring the S/N at 3:1 for LOD and 10:1 for the LOQ in plasma [31]. Both LOD and LOQ were recorded by comparing the S/N of known low concentrations of ART, LFT, PYT, SFD, and MFL with those of blank plasma samples. As is apparent from Table 1, the LOD values for ART, LFT, PYT, SFD, and MFL were 0.1, 0.01, 0.02, 0.05, and 0.04 g/mL, respectively; and the LOQ values were 0.3, 0.03, 0.06, 0.15, and 0.15 g/mL, respectively. 3.2.6 Robustness The matrix of experiments and obtained responses is presented in Supporting Information Table S2. The significant factor effects were graphically identified using Pareto charts at 5% level of significance (Supporting Information Fig. S2). Moreover, none of the factors in chosen range showed significant effect on quality of the separation. As expected, the mobile phase flow rate demonstrated a significant influence on total analysis time (Supporting Information Fig. S2). From Supporting Information Fig. S2, it was confirmed that the method was robust in the investigated domain. However, when the flow rate of the mobile phase was altered there was
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The adequacy of the proposed method for the assay of formulations was tested by analyzing eight commercially available tablets namely, Arte plus, Coartrin, and Arlufe containing ART and LFT; Amalar Forte, Croyodoxin FM, and Malocide containing PYT and SFD; Confal, Mefliam, and MFL containing MFL. Each sample was analyzed in four replicates. Supporting Information Table S4 shows the label claim specified by the manufacturers and those ones obtained according to the developed method. The recoveries achieved for the assay (97.5–103.2%) agreed with the label claim of the manufacturers.
4.1 Advantages of the developed method The method’s higher sensitivity, simplicity, and reliance on simpler HPLC equipment are the main advantages of the developed method. The proposed method has other potential advantages in terms of reduced analysis time (˂10 min) and utilization of simple ternary mobile phase. This is the first isocratic method that allows the chromatographic resolution of all the five antimalarial drugs with UV detection, which allows straightforward application in QC and pharmacokinetic investigations.
5 Concluding remarks This paper presents a simple, rapid, and robust liquid chromatographic method for simultaneous determination of PYT, SFD, ART, LFT, and MFL in pharmaceutical formulations and human plasma. It allows separating five of the most
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counterfeited antimalarials in a single chromatographic run. The developed method is relatively simple, economic, and obtained results demonstrate its accuracy, precision, and specificity. Therefore, it could be successfully employed for routine QC of antimalarial drugs in bulk drugs and in pharmaceutical formulations. In addition, the results obtained for the determination of antimalarials in human plasma validate the pertinence of proposed method in pharmacokinetic investigations and therapeutic drug monitoring.
[14] Onah, J. O., Odeiani, J. E., J. Pharm. Biomed. Anal. 2002, 30, 851–857.
We gratefully acknowledge the support of Drugs Testing Laboratory, Udhagamandalam, Tamil Nadu, India for providing the facility to carry out the research.
[17] Astier, H., Renard, C., Cheminel, V., Soares, O., Mounier, C., Peyron, F., Chaulet, J. F., J. Chromatogr. B Biomed. Sci. Appl. 1997, 698, 217–223.
The authors declare no conflict of interest.
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[13] Debrus, B., Lebrun, P., Kindenge, J. M., Lecomte, F., Ceccato, A., Caliaro, G., Mbay, J. M. T., Boulanger, B., Marini, R. D., Rozet, E., Hubert, P., J. Chromatogr. A 2011, 1218, 5205–5215.
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[18] Bustos, D. G., Lazaro, J. E., Gay, F., Pottier, A., Laracas, C. J., Traore, B., Diquet, B., Trop. Med. Int. Health 2002, 7, 584–591. [19] Green, M. D., Mount, D. L., Nettey, H., J. Chromatogr. B 2002, 767, 159–162. [20] Arayne, M. S., Sultana, N., Siddiqui, F., Naseem, S., Qureshi, F., Med. Chem. Res. 2010, 19, 1043–1054. [21] Sinnaeve, B. A., Decaestecker, T. N., Risha, P. G., Remon, J.-P., Vervaet, C., VanBocxlaer, J. F., J. Chromatogr. A 2005, 1076, 97–102. [22] Sabarinath, S., Singh, R. P., Gupta, R. C., J. Chromatogr. B 2006, 842, 36–42. [23] Lamalle, C., Marini, R. D. E. A., Debrus, B., Lebrun, P., Crommen, J., Hubert, P., Servais, A.-C., Fillet, M., Electrophoresis 2012, 33, 1669–1678. [24] ICH—International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use—Q2 (R1), Validation of Analytical Procedures: Methodology, 1996, London, Complementary Guideline on Methodology incorporated in November 2005. [25] Guidance for Industry, Bioanalytical Method Validation, Center for Drug Evaluation and Research, USFDA, Rockville, MD 2001. [26] Mannemala, S. S., Nagarajan, J. S. K., Biomed. Chromatogr. 2015, 29, 346–352. [27] Zhao, X., Zhao, Y.-L., Liu, X.-M., Han, W., Yu, Z.-G., J. Sep. Sci. 2012, 35, 984–993. [28] Ma, H., You, J., Liu, Y., J. Sep. Sci. 2012, 35, 1539– 1546. [29] Hewala, I. I., Moneeb, M. S., Elmongy, H. A., Wahbi, A.-A. M., Talanta 2014, 130, 506–517. [30] Dejaegher, B., Vander Heyden, Y., J. Chromatogr. A 2007, 1158, 138–157. [31] Valente, A., Bronze, M. R., Bicho, M., Duarte, R., Costa, H. S., J. Sep. Sci. 2012, 35, 3427–3433.
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ISSN 1615-9306 · JSSCCJ 38 (9) 1441–1624 (2015) · Vol. 38 · No. 9 · May 2015 · D 10609
JOURNAL OF
SEPARATION SCIENCE
Methods Chromatography · Electroseparation Applications Biomedicine · Foods · Environment
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Sai Sandeep Mannemala1,2 Janaki Sankarachari Krishnan Nagarajan1 1 Department
of Pharmaceutical Analysis, JSS University, Udhagamandalam, Tamil Nadu, India 2 Department of Pharmacy, Annamalai University, Annamalai Nagar, Tamil Nadu, India Received December 25, 2014 Revised February 3, 2015 Accepted February 5, 2015
Research Article
Development and validation of a generic liquid chromatographic method for the simultaneous determination of five commonly used antimalarial drugs: Application to pharmaceutical formulations and human plasma A simple, sensitive, and rapid liquid chromatographic method was developed and validated using diode array detection for the determination of five commonly used antimalarial drugs in pharmaceutical formulations and in human plasma. Chromatographic separation of antimalarial drugs and internal standard (ibuprofen) was achieved on a C18 column with a mobile phase composed of 10 mM dipotassium orthophosphate at pH 3.0, methanol, and acetonitrile in a ratio of 20:38:42 v/v, at a flow rate of 1 mL/min. The analytes were monitored at 220 nm and separated in ˂10 min. The method was validated for linearity, accuracy, precision, limit of quantification, and robustness. Both intra- and interday precisions (in terms of %RSD) were lower than 3% and accuracy ranged from 98.1 to 104.5%. Extraction recoveries were ࣙ96% in plasma. The limits of quantitation for artemether, lumefantrine, pyrimethamine, sulfadoxine, and mefloquine were 0.3, 0.03, 0.06, 0.15, and 0.15 g/mL in human plasma. Stability under various conditions was also investigated. The method was successfully applied for quantification of antimalarial drugs in marketed formulations and in spiked human plasma. The method can be employed for routine QC purposes and in pharmacokinetic investigations. Keywords: Antimalarial drugs / Bioanalytical methods / Fractional factorial design / High-performance liquid chromatography / Human plasma DOI 10.1002/jssc.201401465
Additional supporting information may be found in the online version of this article at the publisher’s web-site
1 Introduction Malaria is one of the most common infectious diseases and a great public health issue worldwide, predominantly in Africa and South Asia. Although, malaria is preventable and treatable, it continues to cause more than 600 000 deaths per year [1]. The emergence of drug-resistant strains of Plasmodium became foremost complication in control of the disease which threatens the effectiveness of treatment. To combat the infection, combination therapies are highly advocated. The current first-line drug therapy for chloroquine-resistant malaria is sulfadoxine (SFD) and pyrimethamine (PYT) Correspondence: Sai Sandeep Mannemala, Department of Pharmacy, Annamalai University, Annamalai Nagar, Chidambaram, Tamil Nadu 608001, India E-mail: [email protected]
Abbreviations: ART, artemether; IBU, ibuprofen; IS, internal standard; LFT, lumefantrine; MFL, mefloquine; PYT, pyrimethamine; SFD, sulfadoxine
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with mefloquine (MFL) or parallel courses with quinine and doxycycline as second-line of medication [2]. In cases of multidrug resistance, the fixed combination of artemether (ART) and lumefantrine (LFT) is recommended with MFL [3]. One major concern in the treatment of malaria is the production of substandard and counterfeited drugs which pose an immediate threat to public health and undermine malaria control efforts. The counterfeited drugs result in declined therapeutic efficacy, which consecutively risk the drugs to reach the effective therapeutic concentration levels required for elimination of the parasites. About 15% of all antimalarial drugs in circulation worldwide are believed to be counterfeited, with the figures rising to as high as 50% in some parts of Africa and Asia [4]. So, it is indeed important to control the quality of antimalarial drugs. The combination therapy also demands sensitive and selective analytical methods for the simultaneous quantification of these drugs in biological matrices from a pharmaceutical perspective means of understanding pharmacokinetics and therapeutic drug monitoring.
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This requires validated methods that are sensitive, accurate, and precise. ART (Supporting Information Fig. S1a), (3R,5aS,6R,8aS,9R,10S,12R,12aR)-10-methoxy-3,6,9trimethyldecahydro-12H-3,12-epoxy[1,2]dioxepino[4,3-i] 2-benzopyran, is a methyl ethyl derivative of artemisinin, which is highly effective against the blood schizonts of both malarial parasites Plasmodium falciparum and Plasmodium vivax. LFT (Supporting Information Fig. S1b), 2(dibutylamino)-1-[(9Z)-2,7-dichloro-9-(4-chlorobenzylidene)9H-fluoren-4-yl]ethanol, is used to treat acute uncomplicated malaria in combination with ART to improve efficacy in infections acquired in chloroquine-resistant strains and its biological activity is related to class-2 arylamino alcohols. PYT (Supporting Information Fig. S1c), 5-(4-chlorophenyl)6-ethyl-2,4-pyrimidinediamine, is a derivative of diaminopyrimidine, which is used in combination with SFD in the treatment of malaria. SFD (Supporting Information Fig. S1d), 4-amino-N-(5,6-dimethoxy-4-pyrimidinyl)benzene sulfonamide, is a sulfonamide derivative used in the treatment of malaria. MFL hydrochloride (Fig. 1e), (R,S)-2,8[bis(trifluoromethyl)quinolin-4-yl]-(2-piperidyl) methanol, is 2-aryl substituted analogue of quinine, which is used in second-line treatment of chloroquine-resistant P. falciparum malaria. Ibuprofen (IBU; Supporting Information Fig. S1f), (RS)-2-[4-(2-methylpropyl)phenyl]propanoic acid, is a nonsteroidal anti-inflammatory drug and derivative of propionic acid used for relieving pain and fever and reducing inflammation. Several methods have been reported for determination of ART and LFT in pharmaceutical formulations by HPLC coupled with UV detection [5–10] or MS detection [11–13]. Additionally, there are many methods reported for the determination of PYT and SFD in pharmaceutical formulations and biological matrices by UV spectrophotometry [14, 15], by HPLC coupled with UV [16–20] or MS detection [21, 22]. Lamalle et al. [23] developed a generic micellar electrokinectic chromatographic method for the separation of 15 antimalarial drugs. The reported method does not include the determination of LFT and electrophoretic methods cannot be employed for routine QC due to economic considerations. Hodel et al. [11] reported an LC–MS/MS method for the determination of 14 antimalarial drugs and their metabolites. The method employs a complex gradient elution which involves longer equilibration time (ࣈ21 min) and chromatographic run time (16 min). Debrus et al. [13] proposed an innovative HPLC method for screening 19 antimalarial drugs using design of experiments approach. Although, the method uses novel Quality by Design methodology, it employs a complex gradient elution and requires higher run time for the analysis. Moreover, none of the above proposed methods have been employed for determination of antimalarials in pharmaceutical formulations or biological matrices. Together, these studies highlight the demand of a generic HPLC–PDA method which is simple, short, precise, robust, and accurate as well as cost-effective for the analysis of selected antimalarial drugs. Although one patient would not be prescribed all antimalarial C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
drugs under study at once, the proposed method has the advantages of providing suitability for routine clinical investigations, bioequivalence studies, and drug–drug interaction studies which demands detection of more than one analyte in biological matrices. The present study describes the development of a simple, economic, and sensitive HPLC–PDA method for simultaneous determination of ART, LFT, PYT, SFD, and MFL in pharmaceutical preparations and human plasma. Sample pretreatment involved a simple protein precipitation technique. The method was validated and was applied for QC of the formulations and determination in human plasma. To our knowledge, this is the first report of the HPLC–PDA method for the simultaneous determination of the selected antimalarials in formulation and human plasma with UV detection in shorter runtime.
2 Materials and methods 2.1 Chemicals and reagents Pure standards of ART (99.1%) and LFT (99.3%) were generously donated by Medreich Limited (Karnataka, India), PYT (98.6%) and SFD (99.5%) were provided by Nosch labs (Andhra Pradesh, India), MFL (98.9%) and IBU (99.4%; internal standard (IS)) were a generous gift from Micro Labs (Bangalore, India). Acetonitrile of HPLC grade was purchased from Sigma–Aldrich (Bangalore, India), dipotassium orthophosphate and phosphoric acid of AR grade were purchased from SD fine chemicals (Mumbai, India) and Milli-Q water was prepared from Milli-Q Academic, Millipore (Bangalore, India). The tablets Arte plus (Zydus Cadila Healthcare, India), Coartrin (Medreich Saimirra, India), and Arlufe (Neiss Labs, India) containing ART and LFT; Amalar Forte (Micro Labs, India), Croyodoxin FM (Glaxo Smithkline pharmaceuticals, India), and Malocide (Torrent Labs, India) containing PYT and SFD; Confal (Lupin laboratories, India), Mefliam (Cipla, India), and MFL (Emcure Pharmaceuticals, India) containing MEF were purchased from local pharmacy.
2.2 Plasma sampling Drug-free human blood samples were collected from healthy volunteers in tubes containing sodium citrate solution. The samples were centrifuged for 10 min at 1500 rpm for separation of plasma from blood. Subsequently, the samples were transferred to polypropylene tubes and stored at –20⬚C until further use.
2.3 Instrumentation and chromatographic conditions Chromatographic measurements were performed using a Shimadzu HPLC (Shimadzu, Tokyo, Japan) model equipped with Shimadzu LC-10 AT VP solvent delivery module www.jss-journal.com
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Figure 1. Representative chromatograms corresponding to (A) a synthetic mixture containing PYT, SFD, ART, LFT, MFL, and IBU (IS), under optimal conditions; (B) processed blank human plasma; (C) drugs PYT, SFD, ART, LFT, and MFL spiked in human plasma with IBU (IS).
with SPD M-10AVP PDA detector. Data acquisition was performed using Shimadzu Class-VP software. Chromatographic analysis was carried out using Phenomenex Gemini C18 Column (150 × 4.6 mm, 5m; Phenomenex , USA). The mobile phase was a mixture of dipotassium orthophosphate R
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buffer/acetonitrile/methanol in ratio of 20:38:42 v/v adjusted to a pH of 3.0 using phosphoric acid. The mobile phase was filtered through 0.22 m membrane filter (Gelman, India) and degassed ultrasonically for 15 min before use. The flow rate was set at 1 mL/min. A sample volume of 20 L was www.jss-journal.com
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injected for the chromatographic analysis. Based on the obtained isobestic point, the detector was set at 220 nm for the determination of all the analytes.
2.4 Analysis of tablets
ples (limit of quantification (LOQ), low QC, medium QC, and high QC) were prepared in blank human plasma at concentrations of 0.3, 1, 2.5, and 5 g/mL for ART; 0.03, 0.1, 0.25, and 0.5 g/mL for LFT; 0.06, 0.2, 0.5, and 1 g/mL for PYT; 0.15, 0.5, 1.5, and 2.5 g/mL for SFD and MFL. All samples were stored at –70⬚C until further use.
2.4.1 Preparation of stock and standard solutions
2.5.2 Plasma sample preparation
The standard stock solutions of ART, LFT, PYT, SFD, MFL, and IBU at 1000 g/mL were prepared separately by dissolving appropriate amounts of each compound in acetonitrile and methanol mixture, except for LFT in which 2 mL of chloroform is added to ensure its complete solubility. Working standard solutions were prepared by diluting the stock solutions with mobile phase to obtain a concentration range of 10, 15, 20, 25, and 30 g/mL for ART; 20, 40, 60, 80, and 100 g/mL for LFT, PYT, and MFL; 2, 4, 6, 8, and 10 g/mL for SFD.
During the day of analysis, the samples were removed from the deep freezer and were allowed to thaw. 250 L of aliquots were transferred to polypropylene tubes, spiked with 50 L of IBU (5 g/mL) as IS and mixed for 30 s. To the mixture, 300 L of acetonitrile was added and was vortex mixed for 3 min. After centrifugation at 1000 rpm for 10 min, the supernatant was transferred to polypropylene tubes. The collected supernatant was filtered using 0.22 m membrane filter and the filtrate was evaporated to dryness under the stream of nitrogen gas. The residue was reconstituted with 300 L of the mobile phase and a 20-L aliquot was injected into the HPLC system.
2.4.2 Preparation of tablet placebo The analytical placebo solutions were prepared by dissolving the stated excipients in selected tablet formulations in ethanol and were used for validation. 2.4.3 Preparation of tablet sample solutions For assay, 20 tablets containing (ART + LFT), (PYT + SFD), and MFL were weighed and powdered individually. An amount of tablet powder equivalent to 50 mg was weighed and transferred to a 50 mL volumetric flask followed by addition of mobile phase and IS. The mixture was subjected to sonication for 20 min and the solution was made up to the mark with the mobile phase. The solutions were centrifuged at 4000 rpm for 10 min. The clear supernatant was collected and filtered through a 0.22 m membrane filter. An aliquot of 20 L of this solution was used for HPLC analysis.
2.5 Analysis of plasma samples 2.5.1 Preparation of working standard solutions and QC samples The stock solutions of the antimalarial drugs and IS were prepared according to the procedure described in Section 2.4.1. The stock solutions were further diluted five- to 20-fold with methanol and acetonitrile mixture to prepare a series of working standard solutions. Calibration standards were prepared by spiking 20 L of prepared working standard solutions to 230 L human plasma to produce 0.3, 0.6, 0.9, 1.8, 2.4, 3.6, 4.8, and 6 g/mL for ART; 0.03, 0.06, 0.09, 0.18, 0.24, 0.36, 0.48, and 0.6 g/mL for LFT; 0.06, 0.12, 0.18, 0.36, 0.48, 0.72, 0.96, and 1.2 g/mL for PYT; 0.15, 0.3, 0.45, 0.9, 1.2, 1.8, 2.4, and 3 g/mL for SFD and MFL. Four levels of QC sam C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
2.6 Statistical tools Experimental design and Pareto chart analysis was performed by using Statgraphics Centurion XVI, trial version 16.2 (StatPoint Technologies, Warrenton, VA, USA).
2.7 Method validation The developed HPLC–PDA method for the determination of antimalarials in tablets was validated in compliance ICH guidelines [24], while the validation of method for the determination of antimalarials in human plasma was performed by following the USFDA-CDER guidelines [25]. The tested parameters were selectivity, accuracy, precision, linearity, recovery, LOD, LOQ, and stability. 2.7.1 Selectivity and specificity The specificity of the method for determination of antimalarials in tablets was tested by comparing the chromatograms obtained by analyzing the prepared placebo sample with that of the selected antimalarial drugs. Furthermore, the selectivity of the method was evaluated by comparing the peak purity plots obtained for analytes in both sample and standard solutions [24]. The selectivity of the method for determination of antimalarials in human plasma was assessed by analyzing the presence of potential chromatographic interferences of plasma endogenous compounds at the retention times of PYT, SFD, MFL, ART, LFT, and IS. For this, the blank plasma samples obtained from six different subjects was processed and the chromatograms were compared with spiked plasma samples [26]. www.jss-journal.com
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2.7.2 Linearity The linearity of the developed analytical method for tablet matrix was established by analyzing five concentrations each over a range of 5–30 g/mL for ART, 2–10 g/mL for SFD, 10–50 g/mL for MFL, and 20–100 g/mL for LFT and PYT. Six replicates per concentration were injected and chromatograms were recorded. Calibration curves were plotted by using recorded peak area ratios of the drug versus nominal concentration of drug. The acceptance criterion for each back-calculated standard concentration was 100 ± 2% with %RSD lower than 2% [24]. The linearity of the developed bioanalytical method was tested by analyzing eight nonzero calibration standards of 0.3–6 g/mL for ART, 0.03–0.6 g/mL for LFT, 0.06– 1.2 g/mL for PYT, 0.15–3 g/mL for SFD and MFL. Six replicates per concentration were injected and chromatograms were recorded. The acceptance criterion for each backcalculated standard concentration was 100 ± 10% (%RSD ࣘ 10%) except at the LOQ concentration, where the %RSD should not exceed 15% [25]. 2.7.3 Accuracy and precision The accuracy and precision of the analytical method was evaluated in five replicates at three concentration levels 80, 100, and 120% by spiking the known amount of working standard solutions to the tablet placebo. The criteria for the acceptability of the accuracy data are ±2% deviation from the nominal value and for precision within ±2 %RSD [24]. All the samples were analyzed in five replicates on three consecutive days. The accuracy and precision of the proposed bioanalytical method was evaluated at four concentration levels covering the entire linearity range including the LOQ concentration. All the samples were analyzed in five replicates on three consecutive days. The criteria for the acceptability of the accuracy data are ±15% deviation from the nominal value and for precision within ±15 %RSD [25]. 2.7.4 Recovery The absolute recoveries of antimalarial drugs from human plasma were assessed at four concentration levels from 0.3 to 0.5 g/mL for ART, 0.03 to 0.5 g/mL for LFT, 0.06 to 1 g/mL for PYT, 0.15 to 2.5 g/mL for SFD and MFL. The recoveries of the analytes were calculated by comparing the analyte peak area ratios from processed plasma samples with that the corresponding ratios obtained from the aqueous samples at same concentrations [27]. 2.7.5 Stability 2.7.5.1 Tablet solution stability It is necessary to evaluate the stability of both the standard and tablet sample solutions during the entire analytical process. For this, both standard and sample solutions were analyzed C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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over a period of 72 h at 25⬚C. The solutions were considered to be stable if the assay results were within acceptable accuracy (100 ± 2%) and precision (± 2 %RSD). 2.7.5.2 Freeze-thaw stability The stability of antimalarial drugs in human plasma was investigated for three freeze-thaw cycles, at 25⬚C for 3 h and at –70⬚C for four weeks by analyzing prepared QC samples in five replicates each to simulate sample handling and storage time in the freezer before the analysis [28]. The stability was assessed by comparing the mean concentrations of the stability sample with theoretical concentrations. The samples were considered stable if the assay results were within acceptable accuracy (100 ± 10%) [29]. 2.7.6 Robustness testing In this study, the robustness of the method was tested by employing fractional factorial design (24–1 ) [30]. The robustness of the method was tested using the following variables such as concentration of acetonitrile (range: 36–40% v/v), pH (range: 2.9–3.1), strength of buffer (range: 8–12 mM), and flow rate (range: 0.9–1.1 mL/min). Variables that were identified to be critical for the quality of the separation for current analysis such as retention factor of first peak, resolution between critical peak pair, and total analysis time were selected as responses.
3 Results and discussion 3.1 Optimization of chromatographic conditions In this study, a generic approach for the simultaneous determination of five commonly used antimalarial drugs by HPLC was developed. Based on the chemical nature of the analytes, preliminary experiments were conducted by using a C18 column and mobile phase comprising acetonitrile and water in 50:50 ratio. The pH was adjusted to 4 using phosphoric acid. At these conditions, the resolution between PYT and SFD was not satisfactory. Additionally, an increase in the buffer content also showed a remarkable increase in retention of LFT. The optimization of mobile phase composition was based on obtaining the maximum resolution (ࣙ2) and minimum run time ( 0.05) was found between the variances.
3.2 Method validation 3.2.3 Accuracy and precision 3.2.1 Selectivity and specificity The selectivity of the method was assessed by applying the proposed chromatographic procedure to tablets placebo and six different lots of plasma. No interfering peaks were found at the retention times of the selected analytes, indicating no interference from tablet excipients. Additionally, the peak purity angles were within the threshold limits indicating that no peaks were co-eluted with the analytes. Furthermore, from Fig. 1B and C, it can be observed that no endogenous interferences were found in retention times of antimalarials extracted from plasma. 3.2.2 Linearity The obtained regression equations for calibration curves were as follows: y = 0.0017x–0.0013 for ART, y = 0.0034x + 0.001 for LFT, y = 0.0044x + 0.0008 for PYT, y = 0.0016x + 0.0011 for SFD, and y = 0.0081x–0.0462 for MFL. The obtained correlation coefficients for all calibration curves were consistently greater than 0.9990 ± 0.0007 demonstrating good linearity over the entire concentration range. The linearity of the developed bioanalytical method was evaluated using eight nonzero concentrations in six replicates. For determination of antimalarials in human plasma, C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
The accuracy of the developed method was confirmed by computing the percentage recovery of known added amounts of antimalarials at three levels in to a blank matrix (tablet placebo or blank plasma. From Table 2, it can be seen that the recoveries of antimalarials at each level were found to be 98.08– 101.92% from tablet placebo, which lies within the acceptable criteria of the bias ±2%. The intraday and interday precision for developed analytical method was evaluated by analyzing five replicates of PYT, SFD, ART, LFT, and MFL at three different concentration levels and the results are expressed as %RSD (Table 2). The low value of %RSD showed that the method is precise within the acceptance limit of ±2%. From Table 1, it can be seen that both the intraday and interday precision data for the five replicate determinations were within the acceptance range of ±15 %RSD (>3.2%) and accuracy data were within the acceptance range of ±15% (98.1–104.5%) deviation from nominal value. 3.2.4 Recovery From Supporting Information Table S1, it can be seen that the mean recoveries of PYT, SFD, ART, LFT, and MFL were ranged from 96.67 to 102.19, 97.33 to 102.67, 96.69 to 100.46, 96.06 to 103.33, and 98.67 to 101.4%, respectively. The www.jss-journal.com
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Analyte PYT
SFD
ART
LFT
MFL
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a shift in retention times, but this did not affect the peak symmetry and peak areas of the analytes.
Accuracy (%)
Precision (%RSD)
3.2.7 Stability
Concentration (g/mL)
Intraday
Interday
Intraday
Interday
60 80 100 6 8 10 20 25 30 60 80 100 60 80 100
99.25 98.08 98.25 101.39 100.2 99.34 99.41 99.25 98.81 100.86 101.50 98.75 101.07 99.40 98.31
101.6 99.15 98.76 101.49 98.71 99.58 98.17 100.80 99.43 99.18 100.47 99.67 99.83 100.73 101.92
1.41 1.13 0.59 1.24 1.76 0.44 1.19 0.72 1.43 1.27 1.08 0.67 1.01 0.64 0.97
1.67 1.54 1.22 1.03 1.69 0.98 1.54 1.06 1.18 0.69 1.82 1.73 0.24 1.45 1.58
3.2.7.1 Tablet solution stability The tablet and standard solutions were analyzed over a period of 72 h and the results from the stability studies indicate that the peak areas of the analytes remain unaffected by showing %RSD ˂ 1.43 with an accuracy of 100 ± 1.72. No significant degradation was found within the period of evaluation, indicating the solutions were stable. 3.2.7.2 Freeze-thaw stability Three freeze-thaw cycles, short-term stability at 25⬚C for 3 h, and long-term stability at –70⬚C for four weeks showed no significant effect on the stability of QC samples of antimalarials. The results from Supporting Information Table S3 suggest that the antimalarials were stable in plasma when stored in frozen state for the stipulated period of time.
4 Application to commercial tablets recovery of IBU (IS) was calculated and a mean value of 98.4% was obtained. Furthermore, the %RSD values for recoveries of PYT, SFD, ART, LFT, and MFL were very low (ࣘ3.01%), suggesting that the proposed procedure was consistent and efficient in the proposed concentration range. 3.2.5 LOD and quantification The LOD and LOQ were established for all the analytes by measuring the S/N at 3:1 for LOD and 10:1 for the LOQ in plasma [31]. Both LOD and LOQ were recorded by comparing the S/N of known low concentrations of ART, LFT, PYT, SFD, and MFL with those of blank plasma samples. As is apparent from Table 1, the LOD values for ART, LFT, PYT, SFD, and MFL were 0.1, 0.01, 0.02, 0.05, and 0.04 g/mL, respectively; and the LOQ values were 0.3, 0.03, 0.06, 0.15, and 0.15 g/mL, respectively. 3.2.6 Robustness The matrix of experiments and obtained responses is presented in Supporting Information Table S2. The significant factor effects were graphically identified using Pareto charts at 5% level of significance (Supporting Information Fig. S2). Moreover, none of the factors in chosen range showed significant effect on quality of the separation. As expected, the mobile phase flow rate demonstrated a significant influence on total analysis time (Supporting Information Fig. S2). From Supporting Information Fig. S2, it was confirmed that the method was robust in the investigated domain. However, when the flow rate of the mobile phase was altered there was
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The adequacy of the proposed method for the assay of formulations was tested by analyzing eight commercially available tablets namely, Arte plus, Coartrin, and Arlufe containing ART and LFT; Amalar Forte, Croyodoxin FM, and Malocide containing PYT and SFD; Confal, Mefliam, and MFL containing MFL. Each sample was analyzed in four replicates. Supporting Information Table S4 shows the label claim specified by the manufacturers and those ones obtained according to the developed method. The recoveries achieved for the assay (97.5–103.2%) agreed with the label claim of the manufacturers.
4.1 Advantages of the developed method The method’s higher sensitivity, simplicity, and reliance on simpler HPLC equipment are the main advantages of the developed method. The proposed method has other potential advantages in terms of reduced analysis time (˂10 min) and utilization of simple ternary mobile phase. This is the first isocratic method that allows the chromatographic resolution of all the five antimalarial drugs with UV detection, which allows straightforward application in QC and pharmacokinetic investigations.
5 Concluding remarks This paper presents a simple, rapid, and robust liquid chromatographic method for simultaneous determination of PYT, SFD, ART, LFT, and MFL in pharmaceutical formulations and human plasma. It allows separating five of the most
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counterfeited antimalarials in a single chromatographic run. The developed method is relatively simple, economic, and obtained results demonstrate its accuracy, precision, and specificity. Therefore, it could be successfully employed for routine QC of antimalarial drugs in bulk drugs and in pharmaceutical formulations. In addition, the results obtained for the determination of antimalarials in human plasma validate the pertinence of proposed method in pharmacokinetic investigations and therapeutic drug monitoring.
[14] Onah, J. O., Odeiani, J. E., J. Pharm. Biomed. Anal. 2002, 30, 851–857.
We gratefully acknowledge the support of Drugs Testing Laboratory, Udhagamandalam, Tamil Nadu, India for providing the facility to carry out the research.
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The authors declare no conflict of interest.
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