Research Article pubs.acs.org/journal/ascecg
Just-Dip-It (Potentiometric Ion-Selective Electrode): An Innovative Way of Greening Analytical Chemistry Mohamed K. Abd El-Rahman, Hala E. Zaazaa, Norhan Badr ElDin,* and Azza A. Moustafa Analytical Chemistry Department, Faculty of Pharmacy, Cairo University, Kasr-El Aini Street, Cairo, Egypt 11562 S Supporting Information *
ABSTRACT: “Green analytical chemistry” (GAC) is a vital area towards the concept of sustainability. As a consequence of the widespread application of HPLC in drug-related analytical investigations and the resulting contamination of the environment with organic solvents questions have been raised about the toxicity/greenness of HPLC in the ecosystem. Traditional analytical separation technologies yield approximately 50 mL of waste per analytical data point. To this end, the pharmaceutical community continues to search for greener opportunities to markedly reduce the amount of organic waste produced and move from conventional offline separation based methodologies to greener in-line alternatives. In this contribution, we’re adopting a “Just-Dip-It” approach with the ultimate goal of advancing and exploiting the potentiometric sensors to their most effective use in different disciplines of drug development. The unique abilities of these ion-selective electrodes (ISEs) for in-line measurements is the key driver for adoption of GAC principles to improve environmental friendliness of the analytical methods. For a meaningful comparison, this work compares the organic waste resulting from ISEs versus HPLC for degradation kinetics monitoring of active pharmaceutical ingredients (APIs) with respect to the 12 principles of GAC. Ipratropium bromide (IP) was chosen as a hydrolyzable anticholinergic drug, and its degradation kinetics were monitored by the two techniques. The first in-line strategy is attained by dipping a highly integrated IP membrane sensor for continuous monitoring of the hydrolysis kinetics of IP by tracing the emf decline over the time scale. The second off-line strategy utilizes a separation-based chromatographic HPLC method via discontinuous tracking the decrease of IP peak area spectroscopically at 220 nm over time. The advantages and shortcomings of each strategy considering GAC principles are highlighted. The merits of these benign real-time analyzers (ISEs) that can deliver equivalent analytical results as HPLC while significantly reducing solvent consumption/waste generation are described. Finally, an applicable strategy for expansion of the Just-Dip-It approach to different disciplines of drug-related analytical investigations is addressed. KEYWORDS: GAC, In-line monitoring, Off-line monitoring, Ipratropium bromide, Ion-selective electrode, Calix[6]arene, HPLC, Hydrolysis kinetics
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INTRODUCTION The beginnings of green chemistry in the late 1990s, as pointed out by Anastas and Warner, were focused toward green organic synthesis in many fields of chemical industry, principally the pharmaceutical industry.1−9 Rapidly, the term “green analytical chemistry” (GAC) was spawned from green chemistry principles and gained a growing acceptance and wide attention in both industry and academia.10−18 Although activities in the analytical laboratories intrinsically involve small-scale chemical processes, their continuous repetition on a routine basis represents a real risk to humans and the environment. The E-factor of analytical laboratories is 25−100,19 which can be matched with the fine chemicals industry. Within the realm of pharmaceutical drug development, modern and innovative techniques have emerged including HPLC, capillary electrophoresis, NMR, and electrospray MS. Notably, combination of an analytical method with monitoring of the active pharmaceutical ingredients (APIs) concentration © 2016 American Chemical Society
can be distinguished as off-line, at-line, and in-line according to the degree of integration between the two units (analytical technique and analyte matrix). Although HPLC remains the workhorse that offers wonderful advantages to the pharmaceutical analytical society (high-resolution capacity, sensitivity, and specificity); it is, however, the lowest-level combination that confronts various difficulties, such as stopping the reactions before measurements and demanding multiple sample removals and lengthy preparation steps. This intermittent profile results in a slow return of data. At this point, the widespread application of HPLC in drug analysis and the resulting contamination of the environment with organic solvents raise questions about the toxicity/greenness of HPLC in the ecosystem. Approximately, for every analytical data point, HPLC generates Received: January 20, 2016 Revised: April 26, 2016 Published: May 2, 2016 3122
DOI: 10.1021/acssuschemeng.6b00138 ACS Sustainable Chem. Eng. 2016, 4, 3122−3132
Research Article
ACS Sustainable Chemistry & Engineering about 50 mL of chemical waste.20 Even with the current great efforts targeted for greening the HPLC technique through either replacement or reduction in the quantities of the consumed solvent and the generated waste, the new reality may require innovative changes in analytical methodology. The switch to a benign alternative analytical tool that retains reliability, robustness, and operator safety may be reasonable green chemistry solutions especially if it is characterized by being cheaper, minitaturizable, and easily automated. Interestingly, modern electroanalytical chemistry is up to the challenge of investigating new routes fulfilling the requirements of GAC.21,22 The cutting-edge electrochemical sensors are categorized by not only eco-friendliness, but also, being relatively simple, sensitive, energy-saving, and compatible with microfabrication technologies. Among all the electrochemical sensors, potentiometric ISEs has largely developed outside the mainstream of electrochemistry.23,24 Classically, a sensing polymeric membrane cocktail is constructed from PVC-polymer, ion-exchanging salt, plasticizer, and usually a selective receptor (ionophore).25 Thanks to the profound understanding of the underlining theory of ion-selective electrodes (ISEs) and the subsequent insights for optimization of the membrane composition and/or the sensor design,25−27 outstanding progress has been made both in the selectivity and sensitivity of the potentiometric ISEs.28,29 Today, research on ISEs is in a great phase of revival, and truly innovative concepts are being placed forward that open new horizons to the analytical chemistry community. A very interesting aspect of potentiometric ISEs is its dependency on the surface phenomenon, neither column specifications nor optical path length, and therefore minute sample volumes can be measured, imparting miniaturization capability and hence in-line monitoring. This ability to provide real-time measurements is the milestone and the most important feature for offering novel and exciting working strategies closely adhering to the principles of GAC. This study critically probes the opportunities and limitations of two different methodologies with different integration modes to the analytical investigations, namely, ISE and HPLC for both stability indicating determination and tracking the degradation kinetics of easily hydrolyzable ionic drugs. The novelty of the current study comes from performing a side by side comparison under similar experimental conditions and hence the advantages and shortcomings of each technique were directly highlighted with the aim of evaluating the sustainability of the analytical procedure. Ipratropium bromide (IP) (which is a bronchodilator drug used mainly for treatment of asthma and bronchitis) was nominated as an example based on the presence of quaternary nitrogen atom (cationic molecule) and the hydrolytic susceptibility of its ester group. One more reason for its selection is the different lipophilicity of IP and its degradation product which facilitate their chromatographic separations. In this work, the two validated stability indicating methodologies for the determinations of IP in the presence of its alkaline degradants, tropic acid and isopropyl-3-hydroxytropanium bromide, were developed (Scheme 1). Moreover, IP degradation kinetics was studied by the proposed methods and then the kinetic parameters [hydrolysis rate constant (k), t1/2, t90%, and activation energy (Ea)] were estimated.
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Scheme 1. Alkaline Hydrolysis of Ipratropium Bromide41
and acetonitrile were purchased from Sigma-Aldrich (Steinheim, Germany). Polyvinyl chloride (PVC), tetrahydrofuran (THF), and 2-nitrophenyloctyl ether (NPOE) were supplied as reported in the Supporting Information. Atrovent unit dose vials (500 μg/2 mL) were purchased from Boehringer Ingelheim (Egypt). Britton−Robinson buffer (BRB) preparation was described in the Supporting Information. Membrane Sensor Construction. Two PVC membranes contained 33.50% PVC, 66.34% NPOE and NaTPB (0.16%, 5.0 mmol kg−1) for sensor 1, while 33.50% PVC, 65.71% NPOE, 0.16% NaTPB (5.0 mmol kg−1), and 0.63% CX6 (10.0 mmol kg−1) for sensor 2 were prepared separately in two 5 cm Petri dishes. The membrane components totaling 600.0 mg were dissolved in the least amount of THF (6.0 mL). The membranes were left overnight for the evaporation of THF solvent, producing a transparent, flexible and homogeneous matrix. Master membranes of approximately 0.1 mm thickness were obtained. Disks of 5 mm diameter were punched from the master membrane and glued to PVC tubing of 4 mm i.d. with a THF/PVC slurry. Then, equivolume of 0.1 mmol L−1 KCl and 0.1 mmol L−1 IP were used as an electrodes inner solution and 1 mm diameter Ag/AgCl wire was used as an inner reference electrode. Conditioning of the sensors was accomplished by soaking in 0.1 mmol L−1 IP solution for 2 h. A diagram presenting the steps of the procedure is shown in Scheme 2. In-line Potentiometric Measurements. A Jenway digital ion analyzer (Essex, UK) was used for the emf measurements. A doublejunction reference electrode Ag/AgCl |3.0 M KCl∥10% KNO3∥(Orion 900200, MA, USA). Off-line HPLC Measurements. An Agilent HPLC system equipped with a G1310A isocratic pump, a G1314 variable wavelength detector (VWD), and a Rheodyne injector (model 7725I) was used for development of the HPLC method. The measurements were performed using a 5 μm C18 Zorbax TM analytical column (25 cm × 0.46 cm). The mobile phase and the sample were filtered by 0.45 and 0.22 μm Millipore membrane filter, respectively. Moreover, degassing of the mobile phase was completed for 15.0 min in an ultrasonic bath before the use. Construction of Calibration Graphs. Sensors Calibration. Standard solutions of IP in BRB solution of pH 7.0 ranging from 1 × 10−7 to 1 × 10−2 mol L−1 were used for the calibration of the equilibrated sensors. Where, the developed electrodes along with the Ag/AgCl reference electrode were dipped into IP solutions. Under a nonstop stirring, the produced emf values were measured after stabilization to ±1 mV then plotted versus the logarithmic concentration values of IP, and the corresponding regression equation for the linear range was computed. HPLC Method Calibration. Aliquots equivalent to 5−110 μg of IP were transferred separately into 10 mL volumetric flasks and then completed to volume with the mobile phase. The samples were then chromatographed using a mobile phase of; 5 mM diammonium hydrogen phosphate buffer (pH 3.5):methanol (45:55 v/v) with a 1 mL min−1 flow rate and detected at 220.0 nm. A calibration curve was constructed by plotting the peak areas of IP as a function of IP respective concentrations. Determination of IP in Pharmaceutical Preparation. IonSelective Electrode. A volume of 5.0 mL of Atrovent vials was accurately completed to volume (10 mL) with BRB of pH 7 to prepare a solution of 1 mmol L−1. The emf measurements were recorded and the concentration was determined from the calibration plots.
EXPERIMENTAL SECTION
Chemicals and Reagents. Ipratropium bromide (IP), sodium tetraphenyl borate (NaTPB), calix[6]arene (CX6), potassium phosphate (KH2PO4), diammonium hydrogen phosphate, methanol, 3123
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Scheme 2. Schematic Representation of the Procedure Steps from Initial Fabrication of the ISE Membrane to Monitoring of the Degradation Kinetics
HPLC Method. A volume of 2.0 mL of Atrovent vials was accurately completed to volume (10 mL) with the mobile phase. The concentration of this solution was 50.0 μg mL−1. The intensity peak area of dosage form concentration was compared to the calibration curve. pH Effect on the Electrode Response. The pH influence on the sensors response in the range from 2.0 to 12.0 was investigated using BRB through measuring the emf values of 1.0 and 0.1 mmol L−1 IP solutions. Preparation of Degradation Product. The complete procedure for the degradation steps along with the testing for the complete alkaline hydrolysis was discussed in details in the (Supporting Information). Methods Validation. According to the ICH guidelines, validation parameters for the ISE and HPLC methods were performed with respect to linearity range, accuracy, repeatability, robustness, LOD, and LOQ.30 Kinetic Studies. The kinetics studies were performed following the procedures described in our previous work,31,32 and it will be briefly represented in the following; Ion Selective Electrode (In-line Monitoring). The kinetics studies (studying reaction order, effect of buffer pH, and temperature) were investigated: (1) Reaction Order. The influence of the pH on the degradation of IP was determined at 25 °C in BRB of pH 11.0. The solution emf values [Co = 430.4 μg mL−1 (1 mmol L−1)] were measured continuously. (2) pH and Temperature Effect on Hydrolysis Rate. IP degradation was investigated in different BRB solution of pH 11.0 and 12.0 at fixed temperature (25 °C). Then, the temperature effect on IP degradation in BRB of pH 11.0 was determined at three temperature levels 30 ± 5 °C. In each of the above studies; logarithmic percentage of IP residual concentration was studied as a function of time. The degradation parameters [rate constant (k), t1/2, t90%] were estimated. Moreover, an Arrhenius plot was constructed. HPLC (Off-line Monitoring). Off-line HPLC experiments were performed for monitoring IP degradation using a fixed IP concentration [Co = 1.0 mg mL−1] placed separately in different BRB solution of pH 11.0 and 12.0. Then, the temperature effect was studied at three temperature levels 30 ± 5 °C in each of the previously mentioned pH values. Tracking the change in IP concentration over
time was investigated by withdrawing aliquots at 5.0 min intervals. These samples were neutralized and then completed with the mobile phase to a definite volume and chromatographed. The logarithmic percentage of the IP residual concentration was studied as a function of time.
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RESULTS AND DISCUSSION
As a result of the expanded need of pharmaceutical products in everyday to save countless lives especially with the world’s population that approaches 9 billion, there is a great demand for a replacement technology that that can convey similar analytical results as HPLC while substantially reducing both the cost and solvent consumption/waste generation. Consequently, the pharmaceutical analytical community strives toward ‘‘Sustainable Analytical Procedures’’ and continues to search for a benign real-time analyzer that meets the requirements of GAC. To this end, we have recently introduced the first generic approach for in-line monitoring of the degradation kinetics of organic compounds by exploiting the outstanding advantages offered by these potentiometric sensors as a proof-of-concept.31 Subsequently, we have demonstrated the opportunities offered by ISEs in reference to at-line monitoring (UV spectrophotometry) for continuous tracking of the degradation kinetics of pyridostigmine bromide.32 Building on these works and in light of GAC principles, this paper intends to further investigates the opportunities offered by ISEs with respect to off-line techniques (HPLC) for continues tracking of the degradation kinetics of easily hydrolyzable ionic drugs. We have structured our experimental work such that we initially compare the response of an ionophore-free membrane with an ionophore-doped one to select the optimum sensor for determination of IP and monitoring its degradation kinetics. Subsequently, we have performed a stability-indicating HPLC methodology for validation of the obtained results. Finally, we have evaluated the different characteristics of the two analytical methodologies to investigate the opportunities offered by each technique 3124
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calculated and it was found to be 0.23 μmol L−1 (0.099 μgmL−1) for sensor 2 which is approximately 1 order of magnitude lower than sensor 1. For this reason, sensor 2, being more sensitive, was consequently used for IP determination in pharmaceutical formulation and for the degradation kinetic studies. To assess the reversibility of sensor 2, the emf values was measured by altering IP concentrations from 1 μmol and 10 mmol L−1 from the uppermost concentration to the lowermost one. The electrode potentiometric response was found to be reversible and did not exhibit any memory effect, though the required time for attaining equilibrium was somewhat longer than upon reversing the measurements order as presented in Figure S2 (Supporting Information). The potential drift was investigated at the uppermost concentration of IP and the lowermost one in the linearity range (1 μmol L−1 and 10 mmol L−1) over a period of 15 min. The values were 1.24 and 0.25 mV/h, respectively. These substantially small values waive the need for drift correction in the current kinetic study. The electrode reproducibility in terms of slope and standard potential (E0) was studied by carrying out a frequent calibrations by the same membrane intradaily (n = 3) and interdaily over a couple of weeks. The results obtained for the slopes were 59.2 ± 0.5 and 58.0 ± 1.9 mV/dec, respectively, for E0 were 310.4 ± 5.0 and 335.8 ± 25.3 mV, respectively. The obtained results indicated that the constructed ISE exhibited acceptable repeatability; however the minor change in the reproducibility among days makes it appropriate to perform calibrations on a daily basis. Dynamic Response Time. The practical response time was measured at each concentration in the linearity range (1 μmol L−1 to 10 mmol L−1). For sensor 1 and 2, the time required to reach steady emf values (±1 mV) was 11 and 9 s, respectively. The potential−time curve in neutral pH value (where IP molecules are stable) is presented in Figure 2. In addition, it is worth mentioning that the electrode rapid response time enables emf recordings and sensor calibration at the alkaline pH values (11 and 12) prelaunching the hydrolysis of IP molecules as shown in Figure S3 (Supporting Information). This character particularly is one of the critical factors that permit potentiometric tracking of the degradation kinetics successfully. pH and Temperature Effect. For reliable quantitative measurements with the ISEs, a careful investigation of the experimental conditions was performed. Practically, pH values within the range of 3 to 9 are observed to be optimum taking in consideration the target ion chemical form and the performance of sensor. Figure 3 presents the emf-pH profile for 1.0 and 0.1 mmol L−1 IP solutions. At pH values below the stated range, a minor change in emf readings is likely ascribed to H+ ions interference, while at higher values of pH, a consistent decrease in the emf values was observed, which is explained by the hydrolysis of IP molecules. Additionally, the results obtained upon investigation of the temperature effect suggest that the sensors displayed a minor rise in emf values with increasing the temperature in the range of 25.0 to 35.0 °C. However, the obtained calibration plots were parallel, also the response time and slope (mV/decade) do not differ considerably with temperature, demonstrating that the PVC membranes have a practical thermal stability up to 35 °C. Sensor Selectivity. The constructed ISE selectivity coefficient (log Kpot IP.I) was studied with respect to IP hydrolytic products (TA and quaternary alcohol)41 and also versus basically similar QUATs. The calibration plots for neostigmine bromide (NEO), distigmine bromide (DB), pyridostigmine bromide (PYD), and choline (CH) are presented in Figure 4,
aiming to answer the question “which of the two analytical procedures is greener?”. ISE Characteristics Using CX6 Ionophore. The performance of potentiometric sensors doped with sensing ionophore is mainly controlled by the host−guest interaction between the target ion (guest) and the ionophore (host). In the present work, the ionophore calix[6]arene (CX6) was selected based on its exceptional complexation properties to organic quaternary ammonium ions (QUATs).33−39 In order to examine the selective recognition of IP by the CX6 ionophore in the membrane phase, the performance of an ionophore-based ISE for IP (sensor 2) was compared with an ionophore-free ion-exchanger NaTPB (sensor 1) as a control experiment. It was found that the positive IP ion favors the reasonable (7.6 Å) cavity size of CX6 allowing IP to fit well and strongly bonded to the 6 OH-donation sites as shown in Figure S1 (Supporting Information). Figure 1 shows the calibration
Figure 1. Profile of the potential in millivolts versus log concentrations of IP in moles per liter obtained with sensors 1, 2.
plots for both sensors. Compared to sensor 1, the CX6-based sensor shows enhanced selectivity coefficient values and improved Nernstian response slope (discussed below). Table 1 Table 1. Metrological Parameters of the Two Proposed Sensors parameter
sensor 1
sensor 2
slope (mV/decade)a intercept t (mV) LOD (mol L−1)b response time (s) working pH range concentration range (mol L−1) stability (days) correlation coefficient
57.3 284.2 5.1 × 10−6 11 3−9 1 × 10−2 to 5 × 10−6 20 0.9995
59.2 310 2.3 × 10−7 9 3−9 1 × 10−2 to 1 × 10−6 25 0.9997
a Average of five determinations. bLimit of detection (measured by interception of the extrapolated arms of nonresponsive and the Nernstian segments of the calibration plot of Figure 1).
shows the metrological parameters of the proposed sensors (slope, working pH range, quantitation limit, detection limit, linear dynamic range, stability, and response time) evaluated with respect to IUPAC recommendations.40 Notably, reproducible results were displayed by two assemblies of sensors 1 and 2 over a period of 20 and 25 days, respectively. The LOD was 3125
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Figure 2. Potential−time curve of sensor 2 in neutral pH value.
Figure 3. Emf−pH profile for 1.0 and 0.1 mmol L−1 IP solutions.
Figure 4. Calibration plots for IP, PYD, Neo, tropic acid, Ca
2+
, Zn2, DB, Gly, Lys, CH, and Glu for sensor 2.
biased values, the log Kpot IP.I numerical values were not calculated. Also, the selectivity toward some common analytes present in biological samples (glucose, lysine, and glycine) has also been investigated. Remarkably, the ISE preference displayed to IP was credited to the relative hydrophilicity of these biological compounds. It is also important to point out that the hydrolytic products did not show any significant interference with the parent drug. The most probable reasons are the lack of cationic moiety in TA molecule and the absence of phenyl group in the quaternary alcohol that results in decreasing the lipophilicity and partitioning to the organic membrane
and the chemical structures of the tested interferents are shown in Figure S4 (Supporting Information). Obviously, these ions display near-Nernstian response slopes and consequently unbiased log Kpot IP.I were calculated (Table 2). This can be explained by the fact that these QUATs are simply exchanged into the organic membrane phase. Notably, a QUAT ion was anticipated to be complexed by a CX6 ionophore. A justification for the enhanced selectivity toward IP is likely attributable to its more lipophilic character than other QUATs. On the other hand, the inorganic ions (Ca2+, Zn2+) did not display an apparent Nernstian slopes, and to exclude 3126
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ACS Sustainable Chemistry & Engineering Table 2. Logarithmic Selectivity Coefficients, log Kpot IP I, Using the Separate Solutions Method (SSM)
Table 3. Assay Validation Parameters for IP by ISE and HPLC
a log Kpot IP.I
parameter
interferent
sensor 1
sensor 2
pyridostigmine neostigmine distigmine choline
−1.82 −1.33 −2.31 −3.59
−2.01 −1.51 −2.54 −3.93
accuracy (mean ± SD)a precision (RSD %) repeatabilityb intermediate precisionc linearity slope intercept correlation coefficient (r) range robustnessd LOQ LOD
a
Each value is the average of three determination. Calibration curves were obtained by successive dilution, and Nernstian response was confirmed in the concentration range where selectivity was measured.
compared to IP. This high discrimination permits the construction of an IP-ISE for tracking the kinetics of the alkaline hydrolysis process. HPLC Method Development. Several experimental conditions were examined with the aim of optimization of the proposed HPLC method. Importantly, pH of the aqueous portion of mobile phase has a significant role in attaining an optimal separation between IP and TA and it was essential to improve selectivity, peak shape, and retention time. TA contains a carboxylic acid functional group which could be ionized with pH change reflecting the impact in retention time and selectivity. By knowing the pka of TA (4.4),42 it was possible to choose an effective pH for the mobile phase depending on the fact that a buffer is effective at ± one pH units from the pKa of the analyte. Trying different pH values, best separation was attained at pH 3.5. At this pH, TA exists in chiefly ion-suppressed form (un-ionized) in contrast to IP which has fixed positive charge (ionic molecule) allowing considerable resolution. Unlike the previously published reports, we attempt to replace acetonitrile with a greener solvent, methanol, to reduce the HPLC waste stream. The method has been finalized on using a mobile phase of methanol:5 mM diammonium hydrogen phosphate (pH 3.5) (55:45 v/v) with 1 mL min−1 flow rate and UV-detection at 220.0 nm. HPLC chromatogram of IP and its degradation product (TA) reveals a retention time of 3.416 ± 0.02 min for IP and 4.479 ± 0.02 min for TA. A linear relationship between the IP concentrations (5.0−110.0 μg mL−1) and the corresponding peak area under the specified experimental conditions was obtained. Validation Parameters. According to ICH guidelines,30 validation of the ISE and HPLC methods was performed. The linear regression equation of the ISE method is E = 310.0 + 59.2log CIP, with r = 0.9997 (SD = 1.245, n = 5). On the other hand, the linear regression equation for the HPLC method is Y = 9.9652CIP + 48.836, with a correlation coefficient of 0.999 (SD = 0.837, n = 11). The LOD is 1.633 μg mL−1, which is calculated by (3.3σ/S). Here σ is the residuals SD (n = 11) and S is the calibration curve slope. Table 3 presents the validation parameters of the two proposed methods. Clearly, the results obtained demonstrated no significant difference between the ISE and the HPLC methods regarding repeatability, intermediate precision, and accuracy. IP Determination in Pharmaceutical Formulation by Potentiometric ISE and HPLC. The proposed ISE and HPLC methods were successfully employed to assay IP in Atrovent vials and bulk powder. Upon statistical comparison of the obtained results to those of the reported HPLC method,43 no significant difference was observed (Table 4). Obviously, the potentiometric ISE method is straightforward, decrease analysis time, thus reducing the cost per sample.
ISE
HPLC
99.89 ± 0.623
100.54 ± 0.457
0.401 1.476
0.655 1.008
59.2 310 0.9997
9.9652 48.836 0.9999
1 × 10−2−1 × 10−6 mol L−1 0.919 1 × 10−6 mol L−1 2.3 × 10−7 mol L−1
5−110 μg mL−1 0.767 4.949 μg mL−1 1.633 μg mL−1
The accuracy (n = 3), average of three concentrations (5.0 × 10−3, 5.0 × 10−4, and 5.0 × 10−5 mol L−1 for ISE) and (25.0, 55.0, and 75.0 μg mL−1 for HPLC). bThe intraday (n = 3), RSD% of concentrations (1 × 10−3, 1.0 × 10−4, and 1.0 × 10−5 mol L−1 for ISE) and (20.0, 50.0, and 70.0 μg mL−1 for HPLC). cThe interday (n = 3), RSD% of concentrations (1 × 10−3, 1.0 × 10−4, and 1.0 × 10−5 mol L−1 for ISE) and (20.0, 50.0, and 70.0 μgml−1 for HPLC). d Robustness (n = 3), RSD% of the previously determined concentrations under variations in method parameters (mobile phase pH and flow rate for HPLC and pH of the background buffer for ISE). a
Table 4. Determination of IP in Pharmaceutical Formulation Using the Proposed Sensor 2, HPLC, and the Reported Method43 Recovery (%) ± SDa pharmaceutical formulation Atrovent unit dose vials (500 μg/2 mL) t-testc Fc
sensor 2
HPLC method
reported methodb
99.75 ± 0.892
100.12 ± 0.661
99.23 ± 0.853
0.942 (2.306) 1.094 (6.39)
1.844 (2.306) 1.665 (6.39)
Average of five determinations. bHPLC method using C8 column with mobile phase consisting of acetonitrile/phosphate buffer (pH 4; 0.1 mol L−1) (20:80) and UV detection at 210 nm. cThe values in parentheses are the corresponding theoretical values for t and F at P = 0.05.
a
Degradation Kinetics. Studying the Hydrolytic Degradation Reaction of IP by ISE. Experimentally, the membrane sensor was integrated into the degradation medium to trace the decline in emf values second by second, then logarithmic percentage of the residual concentration was studied as a function of time at pH 11 and 12 at three temperature levels 30 ± 5 °C. Clearly, the hydrolysis behavior follows a pseudo-first-order degradation kinetics as shown in Figure S5 (Supporting Information). The corresponding k values were calculated from each line regression equation as shown in Figure 5. It was observed that as the temperature rise, the k values increase with the decrease in t1/2. By comparing the k values obtained at the studied pH values, it was observed that the corresponding k values at pH 12 were larger than those at pH 11. This variation increases with the rise in temperatures; 2.7 fold at 25 ◦ C, 4.6 fold at 30◦ C and 4.3 fold at 35° C as shown in Table 5. From the calculated k values, Arrhenius plot was constructed by establishing a relation between k values and 1/T (kelvin−1) demonstrating the temperature effect 3127
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Figure 5. Pseudo-first-order plots of IP degradation (1 mmol L1) with BRB of pH (a) 11.0 and (b) 12.0 at three temperature levels 30 ± 5 °C using ISE.
HPLC (off-line) are in good agreements in calculation of rate constants and accurate prediction of the profile of degradation under the tested pH and temperature, the two analytical methodologies differ in a number of characteristics. HPLC is undoubtedly the most important method in pharmaceutical analysis of APIs. It is widely used for tracking the chemical degradation kinetics of APIs for anticipating both the expiration dates and the best storage conditions of pharmaceuticals. Interestingly, it is recently coupled with online spectroscopic detectors in the identifying and elucidating the structure of impurities.46 However, from the GAC perspective, HPLC confronts several difficulties; such as operator risk, instrument cost, lengthy steps for sample preparation steps and excessive amount of solvent used/waste generated; for example, in 1 day, the amount of eluent consumed by an individual HPLC instrument, assuming that 40 runs is a typical instrument load. If we assume that for a single run the test and rinse times equal 20 min + 10 min with a 2 mL min−1 flow rate, the eluent consumption per day would be 30 × 0.002 × 40 = 2.4 L. If several HPLC instruments are working, subsequently, the consumption considerations will become an economic and environmental problem. Moreover, HPLC is considered to be one of the most energy-consuming techniques (more than 0.1 kWh per sample). In an attempt to mitigate such constraints, the decision to move into greener in-line electrochemical alternatives like the Just-Dip-It approach almost fulfills the 12 principles of GAC12 as follows: (1) Direct analytical techniques. ISEs can be used for direct monitoring of the APIs within the finished product matrices without the need to sample preparation and extraction; solventless sample preparation. (2) Minimal sample size and number. ISEs depend on a surface phenomenon, thus sample volumes can be very small as well as their ability to extract maximum information from one sample reduces the total number of samples. (3) In situ measurements. ISEs provide an instantaneous collection of analytical data in a cost-effective way and thus enable the move from a batch to a continuous process. (4) Integration of analytical processes. ISEs represent the highest-level combination in which the analytical method is fully integrated with the analytical processes thus minimize reagents usage and energy consumption.
Table 5. Kinetic Data of IP Alkaline Degradation ISE pH of Buffer
temperature (° C)
K (min−1) ± Ua (K = 2)
11.0
25.0 30.0 35.0 25.0 30.0 35.0
0.009 ± 0.00015 0.014 ± 0.00046 0.025 ± 0.00065 0.038 ± 0.000574 0.065 ± 0.00115 0.108 ± 0.002504 HPLC
pH of buffer
temperature (°C)
K (min−1) ± U (K = 2)
11.0
25.0 30.0 35.0 25.0 30.0 35.0
12.0
12.0
a
0.009 0.014 0.025 0.038 0.066 0.109
± ± ± ± ± ±
0.00053 0.00095 0.00142 0.00284 0.00341 0.00554
T1/2 (min)
T90% (min)
79.187 49.330 28.123 18.237 10.671 6.444
11.770 7.332 4.180 2.694 1.558 0.948
T1/2 (min)
T90% (min)
79.187 49.330 28.123 18.237 10.671 6.444
11.770 7.332 4.180 2.694 1.558 0.948
Estimated uncertainty values.
on the hydrolysis rate (Figure 6). Additionally, the activation energy was calculated from the following equation:44 log
Ea ⎛ T2 − T1 ⎞ K2 = ⎟ ⎜ 2.303R ⎝ TT K1 1 2 ⎠
The estimated “Ea” was 18.69 kcal mol−1, which agrees with the literature values of the hydrolytically liable ester group.45 Studying the Hydrolytic Degradation Reaction of IP by HPLC. With the purpose of comparing and validating the ISE results, monitoring the degradation kinetics via HPLC was performed. Likewise, a pseudo-first-order degradation kinetics was observed, as shown in Figure S6 (Supporting Information). This kinetic study was relatively time-consuming due to multiple sampling steps, which form a noncontinuous profile and delaying the estimation of k values. Figure 7 shows a decrease in peak area intensity of 100 μg mL−1 IP and an increase in TA peak intensity. The estimated activation energies from Arrhenius plot were 18.69 ± 1.064 and 18.97 ± 1.650 kcal mol−1 for ISE and HPLC, respectively as shown in Figure 6. Advantages and Shortcomings of Each Strategy Considering GAC Principles. While ISEs (in-line) and 3128
DOI: 10.1021/acssuschemeng.6b00138 ACS Sustainable Chem. Eng. 2016, 4, 3122−3132
Research Article
ACS Sustainable Chemistry & Engineering
Figure 6. Arrhenius Plot for IP hydrolysis with BRB of pH 11.0 and 12.0 using (a) cISE and (b) HPLC.
Figure 7. Off-line monitoring of IP hydrolysis profile in alkaline medium of pH 12 at different time intervals (temp 35 °C).
(6) Avoiding derivatization. Besides the ionic drugs, approximately two-thirds of drugs on the market contain at least one group capable of ionization in a pH range of
(5) Automated and miniaturized methods. ISEs are successfully coupled with recent advances in microfabrication and microelectronics. 3129
DOI: 10.1021/acssuschemeng.6b00138 ACS Sustainable Chem. Eng. 2016, 4, 3122−3132
Research Article
ACS Sustainable Chemistry & Engineering
Future Work. New platforms should be explored to construct extremely low-cost potentiometric sensors to be used in low-income populations keeping in mind mass manufacturing techniques. Moreover, extension of the JustDip-It approach presented here should be explored to different disciplines of drug-related analytical investigations such as (1) In-line monitoring of the dissolution profile of pharmaceutical dosage forms and its impact on the selection and design of the dosage form (2) Monitoring the enzymatic degradation of drug molecules in real-time, e.g., tracking the activity of β-lactamases excreted by various bacteria strains that hydrolytically cleave the β-lactam ring of penicillins and cephalosporins and consequently antibiotic resistance. (3) Quantitative analysis of the APIs in different biological samples which is extremely important in pharmacokinetic studies
2−12 and thus can be directly determined with ISEs without derivatization. (7) Minimal analytical waste. Due to absence of sample preparation step, the generated waste is decreased dramatically, compared to HPLC (10 mL versus 1000 mL in everyday usage). (8) Multianalyte method. Excellently suited for use in “electronic tongues” for simultaneous multicomponent analysis. (9) Minimal use of energy. Unlike HPLC, ISEs are considered to be one of the least energy-consuming laboratory practices and instruments (
