J. Sep. Sci. 2015, 38, 1657–1662
Rafael R. Cunha1 Sandro C. Chaves2 Michelle M. A. C. Ribeiro1 L´ıvia M. F. C. Torres3 ˜ 1 Rodrigo A. A. Munoz Wallans T. P. Dos Santos3 Eduardo M. Richter1 1 Instituto
de Qu´ımica, Universidade Federal de ˆ ˜ Naves de Uberlandia, Av. Joao ´ ˆ Avila, 2121, Uberlandia, MG, Brazil 2 Departamento de Qu´ımica 3 Departamento de Farmacia, ´ Universidade Federal dos Vales do Jequitinhonha e Mucuri, Rodovia MGT 367 - Km 583, 5000, Diamantina, MG, Brazil
Received December 8, 2014 Revised February 23, 2015 Accepted February 24, 2015
Simultaneous determination of caffeine, paracetamol, and ibuprofen in pharmaceutical formulations by high-performance liquid chromatography with UV detection and by capillary electrophoresis with conductivity detection Paracetamol, caffeine and ibuprofen are found in over-the-counter pharmaceutical formulations. In this work, we propose two new methods for simultaneous determination of paracetamol, caffeine and ibuprofen in pharmaceutical formulations. One method is based on high-performance liquid chromatography with diode-array detection and the other on capillary electrophoresis with capacitively coupled contactless conductivity detection. The separation by high-performance liquid chromatography with diode-array detection was achieved on a C18 column (250×4.6 mm2 , 5 m) with a gradient mobile phase comprising 20–100% acetonitrile in 40 mmol L−1 phosphate buffer pH 7.0. The separation by capillary electrophoresis with capacitively coupled contactless conductivity detection was achieved on a fused-silica capillary (40 cm length, 50 m i.d.) using 10 mmol L−1 3, 4-dimethoxycinnamate and 10 mmol L−1 ␤-alanine with pH adjustment to 10.4 with lithium hydroxide as background electrolyte. The determination of all three pharmaceuticals was carried out in 9.6 min by liquid chromatography and in 2.2 min by capillary electrophoresis. Detection limits for caffeine, paracetamol and ibuprofen were 4.4, 0.7, and 3.4 mol L−1 by liquid chromatography and 39, 32, and 49 mol L−1 by capillary electrophoresis, respectively. Recovery values for spiked samples were between 92–107% for both proposed methods. Keywords: Caffeine / Capillary electrophoresis / High-performance liquid chromatography / Ibuprofen / Paracetamol DOI 10.1002/jssc.201401387
Additional supporting information may be found in the online version of this article at the publisher’s web-site
1 Introduction Ibuprofen (IB) is an orally administered, non-steroidal antiinflammatory (NSAI) agent with analgesic and antipyretic properties used extensively in the treatment of arthritis. . Paracetamol (PA) or acetaminophen, is a well-known NSAI drug considered one of the most widely used analgesics in ˜ Naves de Correspondence: Dr. Eduardo Mathias Richter; Av. Joao ´ ˆ ˆ Avila 2121 - Campus Santa Monica - CX 593 - Uberlandia - MG CEP 38408-100, Instituto de Qu´ımica E-mail: [email protected]
Abbreviations: ACN, acetonitrile; CA, caffeine; C4 D, capacitively coupled contactless conductivity detectors; CTAB, cetyltrimethylammonium bromide; DAD, diode array detector; DMX, 3,4 dimethoxycinnamic acid; IB, ibuprofen; NSAI, non-steroidal anti-inflammatory; PA, paracetamol C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
the world, although PA is extremely hepatotoxic . Caffeine (CA) is one of the most common adjuvant analgesic drug also found in many beverages such as coffee and cola drinks. Absorption of PA is accelerated when combined with CA and the pain tolerance is increased [3, 4], meanwhile the antinociceptive effect of IB is significantly potentiated, doubling its effectiveness, by doses of CA [1,5]. The use of PA and IB in the same formulation can be justified due to the supra-additive or synergic analgesic effect produced when PA is combined with NSAI drugs, providing superior analgesia than each drug individually [6, 7]. Several studies in the literature have reported about methods for the simultaneous determination of PA and CA [8–22]. In a smaller scale, some other studies in the literature reported methods for simultaneous determination of PA and IB [23–25] and for IB and CA [26, 27]. However, for the simultaneous determination of CA, PA, and IB, only three methods were reported, two spectroscopic with chemometric www.jss-journal.com
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Table 1. Molecular structure and other physical chemistry characteristics of PA, CA, and IB
C8 H9 NO2 151.16 12.75 9.46 0.91
C8 H10 N4 O2 194.19 20.00 -0.93 -0.55
C13 H18 O2 206.28 0.021 4.85 3.84
Molecular formula Molar mass (g/mol) Solubility (g/L)  pKa  logP 
analysis [28, 29] and one using HPLC with diode array detection (DAD) . The main disadvantage of the reported HPLC method is the time required for each analysis (>30 min), inconvenient for routine analysis of pharmaceutical samples. The absence of fast separations methods using HPLC or CE techniques can be justified by differences in the characteristics of these compounds, such as polarity, solubility, and also pKa values, as seen in Table 1 (from Chemicalize.org, ChemAxon) . The different physicochemical characteristics of IB (logP and solubility) in relation to PA and CA make difficult to obtain a unique procedure for simultaneous determination by HPLC. On the other hand, the CA pKa value hinders its analysis by CZE [32, 33]. Due to its high level of confidence, robustness, reproducibility, and selectivity, HPLC is one of the most known and used separation technique. The United States Pharmacopeia recommends HPLC for QC of drugs in cases of concomitant presence of more than one active ingredient. However, this technique is also known to generate a large amount of waste (mainly organic solvents), and generally requires longer analysis time and high economic investment with maintenance (if compared to CE). On the other hand, CE is a more economic option as a separation technique. Some advantages with regard to HPLC are high analytical rate and insignificant solvents waste. Its drawback still can be considered the high limits of detection due to the small sampling amount . CE with capacitively coupled contactless conductivity detection (C4 D) is a relatively recent electrochemical system that was presented at the end of the 1990s [35–38]. The C4 D detector is based on the difference in conductivity between the BGE and the analyte zone, and therefore all charged species can be detected (universal detector) . Analytes can be detected if they have higher or lower conductivity than the BGE . Due to these characteristics, the CE–C4 D system can be used for quantitative analysis in a wide range of application areas including the simultaneous determination cationic and anionic compounds [33, 39–45] and pharmaceutical analysis [46–49]. In this work, two new and faster methods for the simultaneous determination of CA, PA, and IB are presented. C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
The first one is based on HPLC with diode array detection (HPLC–DAD) and the second one is based on CE with contactless capacitive coupled conductivity detection (CE–C4 D). Both proposed methods can be successfully and reliably used to the QC of pharmaceutical formulations containing CA, PA, and IB. The advantages and disadvantages of both proposed methods are also discussed.
2 Materials and methods 2.1 Reagents and samples Highly pure deionized water (R 18 M⍀cm) obtained from a Millipore Direct-Q3 water purification system (Bedford, MA, USA) was used to prepare all aqueous solutions. Analytical grade phosphoric acid (85% m/v) purchased from Reagen (Rio de Janeiro, Brazil), sodium hydroxide (NaOH) and potassium hydroxide (KOH) from Dinˆamica (Diadema, Brazil), methanol and acetonitrile (ACN) from Tedia (United states), lithium hydroxide (LiOH), cetyltrimethylammonium bromide (CTAB), ␤-alanine, 3,4-dimethoxycinnamic acid (DMX), PA, CA, IB, and ammonium hydroxide from Sigma–Aldrich (St. Louis, United States) were used without further purification. Stock solutions of CA, PA, and IB were freshly prepared just before the experiments in water. KOH was added to the sample solution to assist the solubilization of IB. Solvents as ethanol and methanol can also be used for better solubilization of IB, however, in the presence of these solvents, baseline disturbances were observed in CE analysis. A buffer solution (pH 10.4) containing 10 mmol L−1 DMX, 10 mmol L−1 ␤-alanine, and 15 mmol L−1 LiOH was used as the BGE in CE analysis. Pharmaceutical formulations were obtained from Naturefarm pharmacy, in the city of Montes Claros-MG (Brazil) in two combinations; one with 500 mg PA, 200 mg IB, and 30 mg CA, and another with 325 mg PA, 250 mg IB, and 30 mg CA. In both samples, the following excipients were present: cellulose, corn starch, magnesium stearate, and sodium starch glycolate. For each www.jss-journal.com
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analysis, 20 tablets were powdered in a mortar and an amount of the powder equivalent to one tablet was dissolved in the BGE (CE) or mobile phase (HPLC). The sample and standard solutions were filtered through a membrane filter (pore size of 0.45 mm) before injection in the CE–C4 D or HPLC–DAD system.
(n = 3) were performed by analyzing pharmaceutical samples before and after adding known amounts of CA, PA, and IB. The linearity of the proposed methods was evaluated by analyzing a series of different concentrations (n = 3) of each drug using calibration curves to calculate correlation coefficients (r > 0.99) and intercept values.
2.2 CE measurements
3 Results and discussion
All electropherograms were performed using homemade CE equipment with two compact and high-resolution capacitively coupled contactless conductivity detectors (CE–C4 D) [38, 50, 51]. In this version of detector, the potential (4 Vpp ) and frequency (1.1 MHz) are constant (no optimization is required). More detailed information on the compact and highresolution C4 D can be obtained from a previously published study . The detectors were positioned along the capillary at 10 cm from each end. The fused-silica capillary used in all experiments was 40 cm long (effective lengths of 10 and 30 cm) and 50 m i.d. × 375 m o.d. (Agilent, Folsom, CA, USA). Before use, the capillary was flushed with deionized water for 10 min, 0.1 mol L−1 NaOH for 15 min, again with deionized water for 10 min and finally with BGE for 10 min. The samples were injected hydrodynamically for 1 s at 25 kPa. All experiments were carried out at +25 kV (injection side) with normal EOF.
3.1 Optimization of chromatographic conditions
The HPLC used in this work was a Shimadzu model (Kyoto, Japan) consisting of a DAD, a temperature-controlled column chamber, autosampler, tertiary pump system and fraction collector. The column used was C18 (250×4.6 mm2 , 5 m) from Shimadzu (Kyoto, Japan) with a gradient mobile phase comprising of 20–100% acetonitrile (ACN) in 40 mmol L−1 phosphate buffer pH 7.0 (PB7). The gradient program was: 0–2.5 min, 20% ACN and 80% PB7; 2.5–8.5 min, linear gradient to 100% ACN; 8.5–10.5, 100% of ACN; 10.5–25 min, re-equilibrate column at 20% ACN and 80% PB7 before the next injection. All HPLC analyses were carried out at room temperature (40⬚C) with an injection volume of 10 L and a flow rate of 1.0 mL/min (isocratic), while detection was performed at 220 nm. For HPLC analysis, the stock solutions of the pharmaceuticals were prepared in ACN (HPLC grade) and phosphate buffer pH 7.0 (50:50 v/v).
Simultaneous determination of PA, CA, and IB by HPLC with DAD has been previously proposed .The retention times were 3.9, 11.7, and 32.6 min for PA, CA, and IB, respectively. However, the decrease in retention times would be desirable for methods used in pharmaceutical QC (significant time savings and reduction in waste generation). Initially, tests were performed based on a HPLC method for simultaneous determination of PA and IB described in the literature . In this work, an isocratic mobile phase containing acetonitrile/phosphate buffer pH 7.0 (60:40, v/v) was used. However, in this condition, the coelution of chromatographic peaks of CA and PA was observed. Probably, this was due to the similar polarities of both CA and PA. This result suggests that the separation between CA and PA can only be obtained if the polarity of the mobile phase is increased, as previously shown . However, this condition would cause a high retention time for IB (more apolar than PA and CA). The strategy proposed here to shorten the analysis time and maintaining adequate resolution (Rs 1.5) was the use of gradient elution. The optimized gradient consisted in 20:80 (ACN/phosphate buffer pH 7.0) for 2.5 min, then a linear gradient was started to reach 100% of ACN in 8.5 min, which was kept constant until 10.5 min. Under optimized conditions, the resultant chromatogram for the simultaneous determination of CA, PA, and IB is shown in Fig. 1. The retention times were 4.18± 0.02, 4.63± 0.01, and 9.57± 0.03 min for PA, CA, and IB, respectively. A good peak resolution was observed between PA and CA peaks (Rs = 2.03) and the analysis time for IB was shortened considerably (9.57 min) if compared to the previous work (32.6 min) . Furthermore, it is important to emphasize that the C18 column used in the proposed work was longer (250 mm) than that used in the previous study (125 mm) .
2.4 Analytical method validation procedure
3.2 Optimization of CE conditions
The limits of detection were evaluated based on 3 SB /b, where SB is the SD of the mean value of ten blank measures and b is the slope of the calibration curve by using each separation technique. The precision tests (intra- and inter-day) were evaluated for ten replicate measurements of the same standard solution using the both proposed methods. The recovery tests
To perform the simultaneous determination of CA, PA, and IB by CZE, some difficulties had to be overcome. CA molecules are neutral species in the pH range commonly used in CZE and, to our knowledge, CA determination is only possible by CZE if DMX is used in the BGE composition to generate an anionic complex . Thus, it was required
2.3 HPLC measurements
C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Figure 1. HPLC chromatogram of a standard mixture containing 209 mol L−1 of PA, 15.7 mol L−1 of CA, and 60.7 mol L−1 of IB. Detection at 220 nm; injection volume: 10 L; flow rate: 1.0 mL min−1 . Other operating conditions of HPLC–DAD are given in Section 3.1.
to use 10 mmol L−1 DMX in the BGE. The CA–DMX interaction forms an anionic complex of low-mobility and a peak can be observed immediately after the EOF marker (water tip – C4 D detector) if the CE system is operated in the normal EOF mode. A second required condition is the use of a BGE with pH greater than 9.5 to convert the PA molecules (pKa 9.5) in their anionic form. The third target analyte (IB) presents a pKa value of 4.85 and in solutions with pH > 9.5 (pH of BGE for PA determination), all IB species are present in anionic form. For fast determination of anions by CZE, a usual practice is the reversion of the EOF by the addition of a cationic surfactant to the BGE and, consequently, the anions are analyzed in co-EOF separation mode (faster analysis of anions). However, when the cationic surfactant (CTAB) was added to the BGE, no peak was observed for CA, probably due to the competition of CTAB micelles with DMX, impeding the formation of the anionic complex between CA and DMX [32, 45]. Because of this interference, the cationic surfactant was not used and the counter-EOF separation mode was then adopted for simultaneous determination of CA, PA, and IB. The DMX (pKa 3.9) has no buffer capacity in high pH values (> 9.5) and, therefore, 10 mmol L−1 ␤-alanine (pKa 10.2) was added to the solution for buffer capacity in this pH range. In subsequent tests, three different solutions (KOH, NH4 OH, or LiOH) were tested to adjust the pH of BGEs containing 10 mmol L−1 of DMX and ␤-alanine within the range 9.5–11.0. Better signal-to-background ratio was obtained when LiOH was used in the pH adjustment. Probably, this occurred due to lower mobility of lithium ions relative to potassium and ammonium ions (BGE with lower ionic strength). Consequently, the joule effect was minimized and more stable baseline was observed. In BGEs solutions with pH value ranging from 9.5–10.0, co-migration of both CF and PA peaks was observed. On the other hand, at BGEs with pH values higher than 10.8, a co-migration of both PA and IB C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Figure 2. CE eletropherogram of a standard mixture containing CA, PA, and IB (1 mmol L−1 each). BGE: 10 mmol L−1 DMX, 10 mmol L−1 ␤-alanine, and 15 mmol L−1 LiOH (pH = 10.4); separation voltage: +25 kV (injection side); hydrodynamic injection: 25 kPa for 1.0 s; fused-silica capillary id: 50 m; capillary length: 40 cm; effective length: 30 cm; conductivity detection; *system peak.
peaks was observed. However, at BGE with pH value of 10.4, appropriate resolution was obtained for the three compounds, as can be seen in Fig. 2. In this condition, the migration times were 1.65 ± 0.02, 1.87 ± 0.01, and 2.04 ± 0.03 min for CA, PA, and IB, respectively. In addition, a good resolution was obtained between all peaks (Rs > 4). The influence of buffer concentration was also examined. Studies were carried out with three different concentrations of both DMX and ␤-analine (10, 15, and 20 mmol L−1 ). When the BGE concentration was increased, the time of analysis also increased. This happened due to zeta potential decrease when ionic strength was increased, resulting in slower EOF velocity. To generate an analytical method with higher throughput, a BGE containing 10 mmol L−1 of both DMX and ␤-analine was selected in subsequent studies. The effect of the injection time (0.3, 0.6, 1.0, and 1.5 s at 25 kPa) was also studied to obtain the best performance in the CE experiments. The injection time of 1.0 s yielded the best compromise in terms of efficiency separation, resolution, and S/N.
3.3 Analytical validation of both methods The analytical validation process was carried out to check the performance of both CE and HPLC methods for simultaneous determination of CA, PA, and IB in pharmaceutical samples. In this study, characteristics as linearity, LOD, intra- and inter-day precision, analytical frequency, and accuracy (recovery test) were evaluated. The obtained results are shown in Table 2. For comparison, analytical characteristics from a previously published method (spectrophotometry with chemometric data treatment ) used for simultaneous determination of CA, PA, and IB were also added to Table 2. www.jss-journal.com
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Table 2. Comparison of the analytical characteristics of the proposed methods (CE and HPLC) and a spectrophotometric method previously reported in the literature 
LOD (mol L−1 ) R Linear range (mol L−1 ) Intra-day (%; n = 10) Inter-day (%; n = 3) Recovery (%) Analytical rate (h−1 )
39.2 0.997 300–2000 4.7 5.6 94 27
31.9 0.999 400–8000 1.9 2.0 97 27
48.7 0.998 400–2500 2.3 5.6 107 27
0.68 0.999 6–62 0.42 1.5 92 6
4.40 0.999 83–836 0.52 1.9 96 6
3.38 0.999 24–242 0.51 1.2 103 6
1.04 0.999 5–93 —
0.42 0.999 4–73 —
0.76 0.998 5–116 —
Table 3. Comparison of the results (average ± SD; n = 3) obtained for simultaneous determination of PA, CA, and IB in pharmaceutical samples by HPLC and CE
Label values (mol L−1 )
HPLC-DAD (mol L−1 )
CE-C4 D (mol L−1 )
CA PA IB CA PA IB
11.6 249 73.0 11.6 249 73.0
12.4 ± 0.1 253 ± 2 77.8 ± 0.6 12.6 ± 0.1 251 ± 2 72.9 ± 0.6
11.4 ± 0.4 246 ± 2 74.6 ± 0.4 11.7 ± 0.4 248 ± 3 72.0 ± 0.4
The results presented in Table 2 indicate that both CE and HPLC methods exhibited excellent correlation coefficients (> 0.99), adequate precision according to intra- (RSD < 4.7%; n = 10) and inter-day (RSD < 5.6%; n = 3) studies, and recovery values between 92 and 107%. The linear ranges and limits of detection obtained with the CE–C4 D system are higher than those obtained by HPLC and spectrophotometry. However, this is not a limitation when the objective is the QC of pharmaceutical samples (low detection limits may not be necessary). On the other hand, the CE system (27 injections h−1 ) has great advantages over HPLC (6 injections h−1 ) and spectrophotometry in relation to speed of analysis and minimal solvent/reagent consumption and waste production (green analytical method).
3.5 Application of the proposed methods for analysis of pharmaceutical samples The performance of both CE and HPLC methods has been evaluated through the analysis of pharmaceuticals samples. The obtained results are summarized in Table 3. The results obtained by both proposed methods were in agreement with each other at the 95% confidence level (the calculated t-values from Student’s t-test were smaller than the critical value, 4.303, for two degrees of freedom). The statistical F-test revealed no significant difference in the SDs of the results of both methods at 95% confidence level (in all cases, Fcalculated < Fcritical = 19.0, for two degrees of freedom). C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
4 Concluding remarks In the present work, two new methods (HPLC–DAD and CE–C4 D) for fast and simultaneous determination of PA, CA, and IB in pharmaceutical samples were proposed. Both methods are precise, selective, accurate, and can be used for QC of pharmaceutical samples, which contain the three target compounds. Comparison of both proposed methods has shown that the HPLC–DAD method present better sensibility and lower limits of detection. On the other hand, CE–C4 D method is much faster and consumes less solvent/reagent and, consequently, results in lower environmental impact (green analytical method). Therefore, the CE–C4 D method is more suitable for pharmaceutical samples analysis (considering that low limits of detection are not commonly required in pharmaceutical analyses). In addition, to the best of our knowledge, this is the first time that simultaneous determination of PA, CA and IB using CE–C4 D is reported.
The authors are grateful to CNPq (481683/2013-5; 472465/ 2012-0), FAPEMIG (CEX-APQ-01874-12; CEX-PPM-0050313), Finep and CAPES for financial support. This work is a collaboration research project of members of the Rede Mineira de Qu´ımica (RQ-MG) supported by FAPEMIG.
The authors have declared no conflict of interest. www.jss-journal.com
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