Research article Received: 7 August 2013,
Revised: 23 September 2013,
Accepted: 15 October 2013
Published online in Wiley Online Library: 17 December 2013
(wileyonlinelibrary.com) DOI 10.1002/bmc.3085
Determination of chromatographic dissociation constants of some carbapenem group antibiotics and quantification of these compounds in human urine Ebru Çubuk Demiralaya*, Duygu Koça, Y. Doğan Daldala, Güleren Alsancaka and Sibel A. Ozkanb ABSTRACT: The dissociation constant values (sspKa) of some carbapenem group drugs (ertapenem, meropenem, doripenem) in different percentages of methanol–water binary mixtures (18, 20 and 22%, v/v) were determined from the mobile phase pH dependence of their retention factor. Evaluation of these data was performed using the NLREG program. From calculated pKa values, the aqueous pKa values of these subtances were calculated by different approaches. Moreover, the correlation established between retention factor and the pH of the water–methanol mobile phase was used to determine the optimum separation conditions. In order to validate the optimized conditions, these drugs were studied in human urine. The chromatographic separation was realized using a Gemini NX C18 column (250 × 4.6 mm i.d., 5 μm particles) and UV detector set at 220 and 295 nm. Copyright © 2013 John Wiley & Sons, Ltd. Keywords: Carbapenem group; aqueous pKa values; method optimization; nonlinear regression; internal standard; human urine
Introduction
660
Carbepenems as hydrophilic compounds are β-lactams that contain a fused β-lactam ring and a five-membered ring system that differs from penicillin in being unsaturated and containing a carbon atom instead of the sulfur atom. This class of antibiotics has a broader spectrum of activitiy than do most other β-lactam antibiotics. Meropenem and doripenem have two relevant ionizable functional groups, which means that their acid–base chemistry involves two protons, but in contrast, ertapenem has three relevant ionizable functional groups within the pH ranges of pharmaceutical or physiological importance (Brunton et al., 1996). The acid–base dissociation constant, pKa, is an important parameter in absorption, distribution, metabolism and excretion, formulation development and chromatographic separations (Caliaro and Herbots, 2001). In determination of this parameter, separation-based techniques have been proposed, like capillary electrophoresis and reversed-phase high-performance liquid chromatography (RP-LC). The latter technique has been shown to be a reliable tool for pKa determination. The determination of dissociation constant (pKa) value by liquid chromatography is based on the relationship between the retention factors and the pH values of the mobile phase (Wiczling et al., 2008). Various authors have realized that better models are obtained when the pH in the mobile phase is taken into consideration instead of the aqueous pH of the buffer (Barbosa et al., 2001; Demiralay et al., 2009, 2010; Canbay et al., 2011, 2012). There is only systematic report about pKa values of ertapenem, meropenem and doripenem in different acetonitrile–water binary mixtures using RP-LC (Demiralay et al., 2012). Although methanol– water mobile phases have been used in RP-LC separation
Biomed. Chromatogr. 2014; 28: 660–666
procedures, the pKa values of the studied compounds have not yet been determined in MeOH–water binary mixtures. In the present study, pKa values of these drugs were studied in several MeOH–water mixtures (18, 20 and 22%, v/v), in order to overcome the lack of information related to the acid–base equilibria of this type of compound by means of chromatographic measurements. Although water is not used as mobile phase, the chromatograpic behavior of the compounds must be explained using their pKa values in water. Therefore, aqueous pKa values ww pK a of these compounds were calculated by means of the Yasuda–Shedlovsky equation (Yasuda, 1959) and a linear relationship between the mole fraction of methanol and the ss pK a values. Knowledge of the dissociation constants in hydro-organic media used as mobile phases can be very useful in explaining the chromatographic behavior of analytes. Information about these values is necessary to choose the optimal chromatographic conditions for development of analytical methods for their determination. A search of the literature revealed that a number of reversed-phase liquid chromatography (RP-LC) methods have been developed to determine the individual or several drugs in dosage forms (Ozkan et al., 2001; Mendez et al., 2003) or in
* Correspondence to: E. Çubuk Demiralay, Department of Chemistry, Faculty of Science and Literature, Süleyman Demirel University, 32260 Isparta, Turkey. Email: [email protected] a
Department of Chemistry, Faculty of Science and Literature, Süleyman Demirel University, 32260 Isparta, Turkey
b
Department of Analytical Chemistry, Faculty of Pharmacy, Ankara University, 06100 Tandogan, Ankara, Turkey
Copyright © 2013 John Wiley & Sons, Ltd.
Chromatographic dissociation constants and method validation biological fluids (Gordien et al., 2006; Ikeda et al., 2007, 2008; Legrand et al., 2008). This study describes the combined effect of methanol content and pH of the mobile phase on the retention behavior of ertapenem, meropenem and doripenem. The range of pH values used should be broad enough including the pKa of the analyte. The experimental region was selected in such a way that the retention factors of the antibiotics would stay within the limits 1 < k < 10. RP-LC technique was applied to the simultaneous determination of these antibiotics in human urine. No method for the simultaneous determination of these drugs in human urine has been found in the literature.
Experimental
Preparation of urine calibration standards Separate standard calibration graphs were constructed for each component by plotting the ratio of the peak area of the drug to that of IS against the drug concentration. Calibration standards were prepared by spiking urine with the appropriate amounts of stock solutions. Urine calibration solutions were prepared by adding ertapenem at the concentration 15, 20, 25, 30, 35, 40, 45 and 50 μg/mL and with doripenem and meropenem at the concentrations 20, 25, 30, 35, 40, 45 and 50 μg/mL into 5 mL of blank human urine. The concentration of IS was maintained at a constant level of 6.0 μg/mL. Preparation of the urine samples was carried out as described under preparation of human urine samples. Human urine was spiked with the investigated compounds. The limit of detection (LOD) and limit of quantification (LOQ) for the compounds were determined. The LOD and LOQ were calculated from the following equations using the standard deviation (s) of response and the slope (m) of the corresponding calibration curve (Riley and Rosanske, 1996):
Chemicals and reagents Analytical reagent-grade chemicals were used. Carbapenems used in this work were purchased from different pharmaceutical laboratories: meropenem was from Koçak Pharm. (Istanbul, Turkey), doripenem was from Janssen-Cilag Pharm. (Istanbul, Turkey) and ertapenem was from Merck Sharp & Dohme (Istanbul, Turkey). Methanol (organic modifier), potassium hydrogen phthalate (standard buffer) and sodium hydroxide were obtained from Merck (Darmstadt, Germany). Orthophosphoric acid (minimum 85%) was obtained from Riedel-de Haen (Riedel-de Haen Germany)
Apparatus The chromatographic equipment used consisted of a Shimadzu HPLC system (Shimadzu Technologies, Kyoto, Japan) equipped with a pump (LC-10 AD VP), a UV–vis detector (SPD-10AV VP), a column oven (CTO-10 AC VP) and a degasser system (DGU-14A). A Luna C18 analytical column (250 × 4.6 mm i.d., 5 μM) was used as a stationary phase (Phenomenex®, USA). The measurement of eluent pH is of great importance for the purpose of pKa determination. pH measurements of the mobile phase were carried out with a Mettler Toledo MA 235 pH/ion analyzer (Schwerzenbach, Switzerland) using an M-T combination pH electrode. The pH values of the mobile phases were measured against a 0.05 mol/kg potassium hydrogen phthalate solution as a primary reference standard, dissolved in the appropriate methanol–water medium in accordance with IUPAC rules (Rondinini et al., 1987).
s m
(1)
s : m
(2)
LOD ¼ 3:3
LOQ ¼ 10
Preparation of human urine samples Urine samples from healthly volunteers were used for the validation studies. The urine sample was diluted 5-fold in deionized water. A 2 mL aliquot of diluted urine was transferred to a 110 × 30 mm plastic tube and 3 mL methanol was added to the tube. Stocks of human drug-free urine from healthy volunteers were spiked with different amounts of ertapenem, meropenem and doripenem, while the concentration of the internal standard was fixed at 6.0 μg/mL. The solution was vortexed and the spiked samples were microfiltered through a 0.45 μM filter. A 20 μL volume of each sample was injected into the HPLC system.
Intraday and interday precision Intraday and interday assay precision were determined by simultaneously adding known amounts of each compounds to urine collected from healthy volunteers. Precision was calculated by determining the intra- and inter-day percentage relative standard deviations (RSD).
Recovery studies from human urine samples Chromatographic procedure In this study, the mobile phases used were methanol–water mixtures at three different proportions of organic solvent (18, 20 and 22 %, v/v). At each composition, different pH values were studied, spread over the pH range from 2.5 to 8.0 adjusted by the addition of 1.0 M sodium hydroxide solution containing 25 mM phosphoric acid of the mobile phase. The flow rate of the mobile phase was 1.0 mL/min. The column temperature was kept at 30 °C and the injection volume was 20 μL. The compounds studied had different optimal wavelengths [for meropenem and doripenem 295 nm, for ertapenem and amoxicillin (internal standard, IS) 220 nm].
To keep an additional check on the accuracy of this developed method, recovery experiments were performed by adding the known amount of pure drug to pre-analyzed samples of human urine sample. Blank urine samples were spiked with two different concentrations of each substance and a constant level of an internal standard. These spiked urine samples were processed using the procedures described above for the calibration standards. The percentage recovery was calculated by comparing the concentration obtained from spiked samples with the actual added concentration. After five repeated experiments, the average recovery of these compounds was calculated for each compound.
Results and discussion Preparation of standard solutions
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In LC analysis, stock solutions of the studied compounds (100 μg/mL) were prepared in mobile phase and were kept in the dark and stored under refrigeration at 4 °C to minimize decomposition. All the subsequent dilutions for working standards were prepared with the mobile phase. Sodium hydroxide solution used in pH adjustment was prepared by dilution in water.
The carbapenem group drugs studied here, namely ertapenem, meropenem and doripenem, contain different ionizable fragments such as acidic function (carboxyl group) or basic function (pyrrolidine) (Table 1). These group antibiotics are ampholyte compounds. The chemical structure of doripenem is similar to that of meropenem. Both doripenem and meropenem contain one acidic center, a carboxyl group, and one basic center, a
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662
k H2 Aþ kHA k A k H2 Aþ kHA k A k H3 Aþ k H2 A k HA k A2 3.353 (0.070) 7.285 (0.025) — 3.403 (0.026) 7.819 (0.035) — 3.403 (0.054) 4.099 (0.091) 6.859 (0.208) — pKa1 pKa2 pKa3 pKa1 pKa2 pKa3 pKa1 pKa2 pKa3 pKa4 (0.067) (0.028) (0.038) (0.007) (0.004) (0.037) (0.102) (0.154) (0.054) (0.051)
Copyright © 2013 John Wiley & Sons, Ltd.
Ertapenem
Doripenem
The values in parentheses are the standard deviations.
3.012 0.856 3.793 0.920 0.403 1.621 13.171 8.750 1.959 2.716
(0.016) (0.008) (0.027) (0.006) (0.002) (0.027) (0.178) (0.239) (0.047) (0.027)
pKa1 pKa2 pKa3 pKa1 pKa2 pKa3 pKa1 pKa2 pKa3 pKa4
3.297 (0.012) 7.296 (0.014) — 3.292 (0.017) 7.829 (0.021) — 3.358 (0.097) 4.033 (0.081) 6.907 (0.094) —
k H2 Aþ kHA k A k H2 Aþ kHA k A k H3 Aþ k H2 A k HA k A2
2.113 0.585 2.833 0.683 0.252 1.240 12.003 7.110 0.973 1.962
20% MeOH
k H2 Aþ kHA k A k H2 Aþ kHA k A k H3 Aþ k H2 A k HA k A2
pyrrolidine ring. Two pKa values were determined using the RP-LC method for meropenem and doripenem. A more complex situation occurs with ertapenem. This drug contains two acidic centers (COOH group) and one basic center (pyrrolidin ring). Three pKa values were also determined for this drug using the RP-LC method. In this study, the retention behavior of ertapenem, meropenem and doripenem was investigated by using the combined effect of pH and organic modifier concentration of the eluent. The retention factors were determined for each mobile phase composition and studied pH values. The pKa values and the retention factors of investigated compounds in different methanol ratios calculated by NLREG program (NLREG, 1991) are given in Table 2. One of the most important factors in determining pKa is the reaction medium. The pKa1 values of meropenem and doripenem and the pKa1, pKa2 values of ertapenem obtained in MeOH–water binary mixtures increased and the pKa2 values of meropenem doripenem and pKa3 values of ertapenem decreased with percentage of MeOH. The retention factors were obtained over a pH range of 2.5–8.0 in order to determine the pKa of these antibiotics using the LC method. As an example, in Fig. 1, the experimental data obtained at 20% (v/v) methanol–water binary mixture (k vs pH) are plotted for all compounds, showing the sigmiodal curves. As can be clearly seen, ideal sigmoidal shape was obtained in 20% (v/v) methanol–water binary mixture for ampholyte compounds studied in LC. Evaluation of aqueous dissociation constants ww pK a is an unavoidable requirement in routine drug development. In this
18% MeOH
Amoxicillin
Compounds
Ertapenem
Table 2. The retention factors and the pKa values of investigated compounds in various methanol ratios calculated by NLREG program
Meropenem
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pKa1 pKa2 pKa3 pKa1 pKa2 pKa3 pKa1 pKa2 pKa3 pKa4 1.387 0.342 1.919 0.430 0.179 0.873 10.525 6.279 0.729 1.226
22% MeOH
Doripenem
(0.019) (0.011) (0.004) (0.007) (0.004) (0.036) (0.209) (0.355) (0.059) (0.020)
Structure
Meropenem
Compounds
3.395 (0.031) 7.275 (0.004) — 3.504 (0.046) 7.810 (0.049) — 3.447 (0.135) 4.152 (0.149) 6.809 (0.168) —
Table 1. Structure of ertapenem, meropenem, doripenem and amoxicillin (IS)
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Chromatographic dissociation constants and method validation
Figure 1. Plots of retention factor values of studied compounds vs the pH of the mobile phase for 20% (v/v) of methanol. (A) Doripenem; (B) meropenem; and (C) ertapenem. The theoretical results are indicated as continuous lines and the plotted points are experimental results.
study, sspKa values of studied compounds were determined in the water-rich region of methanol–water mixtures. In addition, two different equations to estimate the ww pK a from the mobile phase pKa values were used. In the first approach, sspKa values were plotted against to methanol mole fraction. The intercepts of these linear equations obtained from this approach were the aqueous pKa values of these compounds (Table 3). In the
Table 3. Aqueous pKa values of studied compounds obtained from different methodologies pK
pKa-X
Yasuda–Shedlovsky
Ertapenem
pKa1 pKa2 pKa3 pKa4 pKa1 pKa2 pKa3 pKa1 pKa2 pKa3
3.015 3.577 7.286 — 2.922 7.377 — 2.476 7.902 —
2.817 3.395 7.196 — 3.102 7.533 — 2.809 8.103 —
Meropenem
Doripenem
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Compounds
second approach, Yasuda–Shedlovsky equation (Yasuda, 1959) was used in order to predict aqueous pKa values from the sspKa values (Table 3). The variation of the pKa values with the mole fraction of methanol was different for the investigated compounds. The pKa1 values of meropenem and doripenem and the pKa1 and pKa2 values of ertapenem increased with the mole fraction of methanol, whereas the pKa2 values of meropenem and doripenem and the pKa3 values of ertapenem decreased with the mole fraction of methanol. The optimization of chromatographic selectivity can be achived by taking into account the dissociation constant and retention factors of the ionized and nonionized forms of the compounds for organic solvent–water binary mixture systems. The aim of this study was to identify the influence of RP-LC conditions on the separation and the retention factor of each analyte and based on a relationship between mobile phase pH and retention factors to determine the optimum separation condition for simultaneous determination of the analytes in human urine. A Luna C18 reversed phase LC column (250 × 4.6 mm i.d., 5 μm) yielded greatly improved efficiencies for studied compounds. Because of the ampholyte character of the studied compounds, their retention is greatly influenced by pH. In this study, the efficiency, selectivity and retention terms were calculated in the
E. Çubuk Demiralay et al. usual way and are summarized in Table 4 for the optimum separation conditions. These can be achieved when the methanol content in the mobile phase is 18 % (v/v) at pH 8.0, in which all solutes are well separated in an analysis time of about 15 min. Under these conditions, the appropriate retention factor (1 < k < 10), selectivity (α ≥ 1.15), resolution (Rs ≥ 1.5) and retention time were obtained. In the other conditions, retention factors of the compounds did not stay within the limits 1 < k < 10. Method validation System suitability tests are used to verify that the resolution and reproducibility of the chromatographic system are adequate for the analysis to be done. According to The United States Pharmacopeia (2000), method , system suitability tests are an integral part of a liquid chromatographic method. System suitability tests were carried out on freshly prepared standard stock solutions of studied compounds. The parameters include tailing factor, retention factor, theoretical plate number, retention time, asymmetry factor, selectivity and RSD (%) of peak height or area for repeated injections. Tailing factors of 1.11, 1.06, 1.12 and 1.09 were obtained for doripenem, ertapenem, meropenem and amoxicillin (IS), respectively. The theoretical plate numbers (N)
were 9505 for doripenem, 8409 for ertapenem, 12418 for meropenem and 11647 for IS. The selectivity factors were 1.947, 1.160 and 1.290 for doripenem, ertapenem and meropenem, respectively. The chromatographic conditions described ensured adequate retention and resolution for all analytes. The retention times of doripenem, IS, ertapenem and meropenem were 6.974, 11.630, 14.050 and 10.479 min. The variation in retention time for five replicate injections of all compound reference solutions gave RSDs of 0.07% for doripenem, 0.46% for ertapenem, 0.33% for meropenem and 0.19% for the IS. The results obtained from the system suitability tests satisfy the United States Pharmacopeia requirements. Using the described analytical method, an optimal resolution of the analytes was achieved. A typical chromatogram is shown in Fig. 2. All examined compounds were well separated in a total duration of 15 min, with good peak resolutions, sharpness and symmetry. Calibration graphs were constructed for all compounds. The method was found to be linear for each compound. Calibration curve parameters are given in Table 5. Intraday and interday assay precisions were evaluated by adding known amounts of doripenem, ertapenem and meropenem into urine. At spiking levels of different concentrations the precision (in terms of RSD) was found to be
Revised: 23 September 2013,
Accepted: 15 October 2013
Published online in Wiley Online Library: 17 December 2013
(wileyonlinelibrary.com) DOI 10.1002/bmc.3085
Determination of chromatographic dissociation constants of some carbapenem group antibiotics and quantification of these compounds in human urine Ebru Çubuk Demiralaya*, Duygu Koça, Y. Doğan Daldala, Güleren Alsancaka and Sibel A. Ozkanb ABSTRACT: The dissociation constant values (sspKa) of some carbapenem group drugs (ertapenem, meropenem, doripenem) in different percentages of methanol–water binary mixtures (18, 20 and 22%, v/v) were determined from the mobile phase pH dependence of their retention factor. Evaluation of these data was performed using the NLREG program. From calculated pKa values, the aqueous pKa values of these subtances were calculated by different approaches. Moreover, the correlation established between retention factor and the pH of the water–methanol mobile phase was used to determine the optimum separation conditions. In order to validate the optimized conditions, these drugs were studied in human urine. The chromatographic separation was realized using a Gemini NX C18 column (250 × 4.6 mm i.d., 5 μm particles) and UV detector set at 220 and 295 nm. Copyright © 2013 John Wiley & Sons, Ltd. Keywords: Carbapenem group; aqueous pKa values; method optimization; nonlinear regression; internal standard; human urine
Introduction
660
Carbepenems as hydrophilic compounds are β-lactams that contain a fused β-lactam ring and a five-membered ring system that differs from penicillin in being unsaturated and containing a carbon atom instead of the sulfur atom. This class of antibiotics has a broader spectrum of activitiy than do most other β-lactam antibiotics. Meropenem and doripenem have two relevant ionizable functional groups, which means that their acid–base chemistry involves two protons, but in contrast, ertapenem has three relevant ionizable functional groups within the pH ranges of pharmaceutical or physiological importance (Brunton et al., 1996). The acid–base dissociation constant, pKa, is an important parameter in absorption, distribution, metabolism and excretion, formulation development and chromatographic separations (Caliaro and Herbots, 2001). In determination of this parameter, separation-based techniques have been proposed, like capillary electrophoresis and reversed-phase high-performance liquid chromatography (RP-LC). The latter technique has been shown to be a reliable tool for pKa determination. The determination of dissociation constant (pKa) value by liquid chromatography is based on the relationship between the retention factors and the pH values of the mobile phase (Wiczling et al., 2008). Various authors have realized that better models are obtained when the pH in the mobile phase is taken into consideration instead of the aqueous pH of the buffer (Barbosa et al., 2001; Demiralay et al., 2009, 2010; Canbay et al., 2011, 2012). There is only systematic report about pKa values of ertapenem, meropenem and doripenem in different acetonitrile–water binary mixtures using RP-LC (Demiralay et al., 2012). Although methanol– water mobile phases have been used in RP-LC separation
Biomed. Chromatogr. 2014; 28: 660–666
procedures, the pKa values of the studied compounds have not yet been determined in MeOH–water binary mixtures. In the present study, pKa values of these drugs were studied in several MeOH–water mixtures (18, 20 and 22%, v/v), in order to overcome the lack of information related to the acid–base equilibria of this type of compound by means of chromatographic measurements. Although water is not used as mobile phase, the chromatograpic behavior of the compounds must be explained using their pKa values in water. Therefore, aqueous pKa values ww pK a of these compounds were calculated by means of the Yasuda–Shedlovsky equation (Yasuda, 1959) and a linear relationship between the mole fraction of methanol and the ss pK a values. Knowledge of the dissociation constants in hydro-organic media used as mobile phases can be very useful in explaining the chromatographic behavior of analytes. Information about these values is necessary to choose the optimal chromatographic conditions for development of analytical methods for their determination. A search of the literature revealed that a number of reversed-phase liquid chromatography (RP-LC) methods have been developed to determine the individual or several drugs in dosage forms (Ozkan et al., 2001; Mendez et al., 2003) or in
* Correspondence to: E. Çubuk Demiralay, Department of Chemistry, Faculty of Science and Literature, Süleyman Demirel University, 32260 Isparta, Turkey. Email: [email protected] a
Department of Chemistry, Faculty of Science and Literature, Süleyman Demirel University, 32260 Isparta, Turkey
b
Department of Analytical Chemistry, Faculty of Pharmacy, Ankara University, 06100 Tandogan, Ankara, Turkey
Copyright © 2013 John Wiley & Sons, Ltd.
Chromatographic dissociation constants and method validation biological fluids (Gordien et al., 2006; Ikeda et al., 2007, 2008; Legrand et al., 2008). This study describes the combined effect of methanol content and pH of the mobile phase on the retention behavior of ertapenem, meropenem and doripenem. The range of pH values used should be broad enough including the pKa of the analyte. The experimental region was selected in such a way that the retention factors of the antibiotics would stay within the limits 1 < k < 10. RP-LC technique was applied to the simultaneous determination of these antibiotics in human urine. No method for the simultaneous determination of these drugs in human urine has been found in the literature.
Experimental
Preparation of urine calibration standards Separate standard calibration graphs were constructed for each component by plotting the ratio of the peak area of the drug to that of IS against the drug concentration. Calibration standards were prepared by spiking urine with the appropriate amounts of stock solutions. Urine calibration solutions were prepared by adding ertapenem at the concentration 15, 20, 25, 30, 35, 40, 45 and 50 μg/mL and with doripenem and meropenem at the concentrations 20, 25, 30, 35, 40, 45 and 50 μg/mL into 5 mL of blank human urine. The concentration of IS was maintained at a constant level of 6.0 μg/mL. Preparation of the urine samples was carried out as described under preparation of human urine samples. Human urine was spiked with the investigated compounds. The limit of detection (LOD) and limit of quantification (LOQ) for the compounds were determined. The LOD and LOQ were calculated from the following equations using the standard deviation (s) of response and the slope (m) of the corresponding calibration curve (Riley and Rosanske, 1996):
Chemicals and reagents Analytical reagent-grade chemicals were used. Carbapenems used in this work were purchased from different pharmaceutical laboratories: meropenem was from Koçak Pharm. (Istanbul, Turkey), doripenem was from Janssen-Cilag Pharm. (Istanbul, Turkey) and ertapenem was from Merck Sharp & Dohme (Istanbul, Turkey). Methanol (organic modifier), potassium hydrogen phthalate (standard buffer) and sodium hydroxide were obtained from Merck (Darmstadt, Germany). Orthophosphoric acid (minimum 85%) was obtained from Riedel-de Haen (Riedel-de Haen Germany)
Apparatus The chromatographic equipment used consisted of a Shimadzu HPLC system (Shimadzu Technologies, Kyoto, Japan) equipped with a pump (LC-10 AD VP), a UV–vis detector (SPD-10AV VP), a column oven (CTO-10 AC VP) and a degasser system (DGU-14A). A Luna C18 analytical column (250 × 4.6 mm i.d., 5 μM) was used as a stationary phase (Phenomenex®, USA). The measurement of eluent pH is of great importance for the purpose of pKa determination. pH measurements of the mobile phase were carried out with a Mettler Toledo MA 235 pH/ion analyzer (Schwerzenbach, Switzerland) using an M-T combination pH electrode. The pH values of the mobile phases were measured against a 0.05 mol/kg potassium hydrogen phthalate solution as a primary reference standard, dissolved in the appropriate methanol–water medium in accordance with IUPAC rules (Rondinini et al., 1987).
s m
(1)
s : m
(2)
LOD ¼ 3:3
LOQ ¼ 10
Preparation of human urine samples Urine samples from healthly volunteers were used for the validation studies. The urine sample was diluted 5-fold in deionized water. A 2 mL aliquot of diluted urine was transferred to a 110 × 30 mm plastic tube and 3 mL methanol was added to the tube. Stocks of human drug-free urine from healthy volunteers were spiked with different amounts of ertapenem, meropenem and doripenem, while the concentration of the internal standard was fixed at 6.0 μg/mL. The solution was vortexed and the spiked samples were microfiltered through a 0.45 μM filter. A 20 μL volume of each sample was injected into the HPLC system.
Intraday and interday precision Intraday and interday assay precision were determined by simultaneously adding known amounts of each compounds to urine collected from healthy volunteers. Precision was calculated by determining the intra- and inter-day percentage relative standard deviations (RSD).
Recovery studies from human urine samples Chromatographic procedure In this study, the mobile phases used were methanol–water mixtures at three different proportions of organic solvent (18, 20 and 22 %, v/v). At each composition, different pH values were studied, spread over the pH range from 2.5 to 8.0 adjusted by the addition of 1.0 M sodium hydroxide solution containing 25 mM phosphoric acid of the mobile phase. The flow rate of the mobile phase was 1.0 mL/min. The column temperature was kept at 30 °C and the injection volume was 20 μL. The compounds studied had different optimal wavelengths [for meropenem and doripenem 295 nm, for ertapenem and amoxicillin (internal standard, IS) 220 nm].
To keep an additional check on the accuracy of this developed method, recovery experiments were performed by adding the known amount of pure drug to pre-analyzed samples of human urine sample. Blank urine samples were spiked with two different concentrations of each substance and a constant level of an internal standard. These spiked urine samples were processed using the procedures described above for the calibration standards. The percentage recovery was calculated by comparing the concentration obtained from spiked samples with the actual added concentration. After five repeated experiments, the average recovery of these compounds was calculated for each compound.
Results and discussion Preparation of standard solutions
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In LC analysis, stock solutions of the studied compounds (100 μg/mL) were prepared in mobile phase and were kept in the dark and stored under refrigeration at 4 °C to minimize decomposition. All the subsequent dilutions for working standards were prepared with the mobile phase. Sodium hydroxide solution used in pH adjustment was prepared by dilution in water.
The carbapenem group drugs studied here, namely ertapenem, meropenem and doripenem, contain different ionizable fragments such as acidic function (carboxyl group) or basic function (pyrrolidine) (Table 1). These group antibiotics are ampholyte compounds. The chemical structure of doripenem is similar to that of meropenem. Both doripenem and meropenem contain one acidic center, a carboxyl group, and one basic center, a
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k H2 Aþ kHA k A k H2 Aþ kHA k A k H3 Aþ k H2 A k HA k A2 3.353 (0.070) 7.285 (0.025) — 3.403 (0.026) 7.819 (0.035) — 3.403 (0.054) 4.099 (0.091) 6.859 (0.208) — pKa1 pKa2 pKa3 pKa1 pKa2 pKa3 pKa1 pKa2 pKa3 pKa4 (0.067) (0.028) (0.038) (0.007) (0.004) (0.037) (0.102) (0.154) (0.054) (0.051)
Copyright © 2013 John Wiley & Sons, Ltd.
Ertapenem
Doripenem
The values in parentheses are the standard deviations.
3.012 0.856 3.793 0.920 0.403 1.621 13.171 8.750 1.959 2.716
(0.016) (0.008) (0.027) (0.006) (0.002) (0.027) (0.178) (0.239) (0.047) (0.027)
pKa1 pKa2 pKa3 pKa1 pKa2 pKa3 pKa1 pKa2 pKa3 pKa4
3.297 (0.012) 7.296 (0.014) — 3.292 (0.017) 7.829 (0.021) — 3.358 (0.097) 4.033 (0.081) 6.907 (0.094) —
k H2 Aþ kHA k A k H2 Aþ kHA k A k H3 Aþ k H2 A k HA k A2
2.113 0.585 2.833 0.683 0.252 1.240 12.003 7.110 0.973 1.962
20% MeOH
k H2 Aþ kHA k A k H2 Aþ kHA k A k H3 Aþ k H2 A k HA k A2
pyrrolidine ring. Two pKa values were determined using the RP-LC method for meropenem and doripenem. A more complex situation occurs with ertapenem. This drug contains two acidic centers (COOH group) and one basic center (pyrrolidin ring). Three pKa values were also determined for this drug using the RP-LC method. In this study, the retention behavior of ertapenem, meropenem and doripenem was investigated by using the combined effect of pH and organic modifier concentration of the eluent. The retention factors were determined for each mobile phase composition and studied pH values. The pKa values and the retention factors of investigated compounds in different methanol ratios calculated by NLREG program (NLREG, 1991) are given in Table 2. One of the most important factors in determining pKa is the reaction medium. The pKa1 values of meropenem and doripenem and the pKa1, pKa2 values of ertapenem obtained in MeOH–water binary mixtures increased and the pKa2 values of meropenem doripenem and pKa3 values of ertapenem decreased with percentage of MeOH. The retention factors were obtained over a pH range of 2.5–8.0 in order to determine the pKa of these antibiotics using the LC method. As an example, in Fig. 1, the experimental data obtained at 20% (v/v) methanol–water binary mixture (k vs pH) are plotted for all compounds, showing the sigmiodal curves. As can be clearly seen, ideal sigmoidal shape was obtained in 20% (v/v) methanol–water binary mixture for ampholyte compounds studied in LC. Evaluation of aqueous dissociation constants ww pK a is an unavoidable requirement in routine drug development. In this
18% MeOH
Amoxicillin
Compounds
Ertapenem
Table 2. The retention factors and the pKa values of investigated compounds in various methanol ratios calculated by NLREG program
Meropenem
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pKa1 pKa2 pKa3 pKa1 pKa2 pKa3 pKa1 pKa2 pKa3 pKa4 1.387 0.342 1.919 0.430 0.179 0.873 10.525 6.279 0.729 1.226
22% MeOH
Doripenem
(0.019) (0.011) (0.004) (0.007) (0.004) (0.036) (0.209) (0.355) (0.059) (0.020)
Structure
Meropenem
Compounds
3.395 (0.031) 7.275 (0.004) — 3.504 (0.046) 7.810 (0.049) — 3.447 (0.135) 4.152 (0.149) 6.809 (0.168) —
Table 1. Structure of ertapenem, meropenem, doripenem and amoxicillin (IS)
Biomed. Chromatogr. 2014; 28: 660–666
Chromatographic dissociation constants and method validation
Figure 1. Plots of retention factor values of studied compounds vs the pH of the mobile phase for 20% (v/v) of methanol. (A) Doripenem; (B) meropenem; and (C) ertapenem. The theoretical results are indicated as continuous lines and the plotted points are experimental results.
study, sspKa values of studied compounds were determined in the water-rich region of methanol–water mixtures. In addition, two different equations to estimate the ww pK a from the mobile phase pKa values were used. In the first approach, sspKa values were plotted against to methanol mole fraction. The intercepts of these linear equations obtained from this approach were the aqueous pKa values of these compounds (Table 3). In the
Table 3. Aqueous pKa values of studied compounds obtained from different methodologies pK
pKa-X
Yasuda–Shedlovsky
Ertapenem
pKa1 pKa2 pKa3 pKa4 pKa1 pKa2 pKa3 pKa1 pKa2 pKa3
3.015 3.577 7.286 — 2.922 7.377 — 2.476 7.902 —
2.817 3.395 7.196 — 3.102 7.533 — 2.809 8.103 —
Meropenem
Doripenem
Biomed. Chromatogr. 2014; 28: 660–666
Copyright © 2013 John Wiley & Sons, Ltd.
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663
Compounds
second approach, Yasuda–Shedlovsky equation (Yasuda, 1959) was used in order to predict aqueous pKa values from the sspKa values (Table 3). The variation of the pKa values with the mole fraction of methanol was different for the investigated compounds. The pKa1 values of meropenem and doripenem and the pKa1 and pKa2 values of ertapenem increased with the mole fraction of methanol, whereas the pKa2 values of meropenem and doripenem and the pKa3 values of ertapenem decreased with the mole fraction of methanol. The optimization of chromatographic selectivity can be achived by taking into account the dissociation constant and retention factors of the ionized and nonionized forms of the compounds for organic solvent–water binary mixture systems. The aim of this study was to identify the influence of RP-LC conditions on the separation and the retention factor of each analyte and based on a relationship between mobile phase pH and retention factors to determine the optimum separation condition for simultaneous determination of the analytes in human urine. A Luna C18 reversed phase LC column (250 × 4.6 mm i.d., 5 μm) yielded greatly improved efficiencies for studied compounds. Because of the ampholyte character of the studied compounds, their retention is greatly influenced by pH. In this study, the efficiency, selectivity and retention terms were calculated in the
E. Çubuk Demiralay et al. usual way and are summarized in Table 4 for the optimum separation conditions. These can be achieved when the methanol content in the mobile phase is 18 % (v/v) at pH 8.0, in which all solutes are well separated in an analysis time of about 15 min. Under these conditions, the appropriate retention factor (1 < k < 10), selectivity (α ≥ 1.15), resolution (Rs ≥ 1.5) and retention time were obtained. In the other conditions, retention factors of the compounds did not stay within the limits 1 < k < 10. Method validation System suitability tests are used to verify that the resolution and reproducibility of the chromatographic system are adequate for the analysis to be done. According to The United States Pharmacopeia (2000), method , system suitability tests are an integral part of a liquid chromatographic method. System suitability tests were carried out on freshly prepared standard stock solutions of studied compounds. The parameters include tailing factor, retention factor, theoretical plate number, retention time, asymmetry factor, selectivity and RSD (%) of peak height or area for repeated injections. Tailing factors of 1.11, 1.06, 1.12 and 1.09 were obtained for doripenem, ertapenem, meropenem and amoxicillin (IS), respectively. The theoretical plate numbers (N)
were 9505 for doripenem, 8409 for ertapenem, 12418 for meropenem and 11647 for IS. The selectivity factors were 1.947, 1.160 and 1.290 for doripenem, ertapenem and meropenem, respectively. The chromatographic conditions described ensured adequate retention and resolution for all analytes. The retention times of doripenem, IS, ertapenem and meropenem were 6.974, 11.630, 14.050 and 10.479 min. The variation in retention time for five replicate injections of all compound reference solutions gave RSDs of 0.07% for doripenem, 0.46% for ertapenem, 0.33% for meropenem and 0.19% for the IS. The results obtained from the system suitability tests satisfy the United States Pharmacopeia requirements. Using the described analytical method, an optimal resolution of the analytes was achieved. A typical chromatogram is shown in Fig. 2. All examined compounds were well separated in a total duration of 15 min, with good peak resolutions, sharpness and symmetry. Calibration graphs were constructed for all compounds. The method was found to be linear for each compound. Calibration curve parameters are given in Table 5. Intraday and interday assay precisions were evaluated by adding known amounts of doripenem, ertapenem and meropenem into urine. At spiking levels of different concentrations the precision (in terms of RSD) was found to be
