Recent advances in the capillary electrophoresis analysis of antibiotics with capacitively coupled contactless conductivity detection

Journal of Pharmaceutical and Biomedical Analysis 158 (2018) 405–415

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Review

Recent advances in the capillary electrophoresis analysis of antibiotics with capacitively coupled contactless conductivity detection Prasanta Paul a , Cari Sänger-van de Griend b,c , Erwin Adams a , Ann Van Schepdael a,∗ a KU Leuven - University of Leuven, Pharmaceutical Analysis, Department of Pharmaceutical and Pharmacological Sciences, O&N2, PB 923, Herestraat 49, Leuven, 3000, Belgium b Department of Medicinal chemistry, Uppsala University, Husargatan 3, Uppsala, 751 23, Sweden c Kantisto BV, Callenburglaan 22, Baarn, 3742 MV, The Netherlands

a r t i c l e

i n f o

Article history: Received 12 May 2018 Received in revised form 18 June 2018 Accepted 19 June 2018 Available online 20 June 2018 Keywords: Capillary electrophoresis Capacitively coupled contactless conductivity Portable devices Antibiotic Review

a b s t r a c t This review describes briefly the high rate of counterfeiting of antimicrobial drugs with focus upon its immediate health consequences. The major part of this review encompasses accounts of the improvements achieved in the domain of miniaturization of capillary electrophoresis with capacitively coupled contactless conductivity detection (CE-C4 D). The application of this principle into the development of portable devices as well as its application to counter the health-system-crippling phenomenon of counterfeit antibiotic formulations, are discussed in the context of developing countries. © 2018 Elsevier B.V. All rights reserved.

Contents 1. 2.

3.

4. 5. 6.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 406 CE with conductivity detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 406 2.1. CE - capacitively coupled contactless conductivity detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 406 2.2. Principle of C4 D . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 407 Overview of low cost portable electromigration devices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 407 3.1. Portable devices to fight counterfeit drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 407 3.2. CE miniaturization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 408 3.2.1. Non-chip based CE-C4 D . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 408 3.2.2. Chip-based (microfluidic) CE-C4 D . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 410 Overview of CE-C4 D application in antibiotic determination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 411 Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 413 Future prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 413 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 413

Abbreviations: WHO, World Health Organization; UNODC, United Nations Office on Drugs and Crime; CD, conductivity detection; C4 D, capacitively coupled contactless conductivity detection; AC-DC, alternating current to direct current; LIF, laser induced fluorescence; BGE, background electrolyte; Qb D, quality by design; MES, 2-(Nmorpholino) ethane sulfonic acid; CTAB, cetyltrimethylammonium bromide; TRIS, tris(hydroxymethyl)aminomethane; TAPS, tris(methylamino)propanesulfonic acid; EOF, electroosmotic flow; BIA, batch injection analysis. ∗ Corresponding author. E-mail address: [email protected] (A. Van Schepdael). https://doi.org/10.1016/j.jpba.2018.06.033 0731-7085/© 2018 Elsevier B.V. All rights reserved.

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1. Introduction Counterfeit and substandard medicines are two phenomena which have been studied extensively in the past. They are prevalent, not only in developing countries, but increasingly becoming ubiquitous in the developed countries as well. The spread of fake drugs is a multifactorial process with significant contributions stemming from diverse sources, ranging from the unawareness and negligence of the patient to the corruption inflicted government agencies [1]. Additionally, internet-based on-line purchase of medicines and fitness products by the patient is contributing to the growing cases of falsified drugs in industrialized countries [2,3]. A survey suggested that half of the on-line purchased drugs are counterfeit [1]. Though drug falsification accounts for 1% of the pharmaceutical market in the developed countries, the reported cases have been steadily increasing in Europe and the US [4]. Published data report the magnitude of drug falsification, ranging from 1 to 30% of the marketed drugs. The scenario in the middle and low-income countries is even worse. Several reports suggested a surprisingly high level of drug counterfeiting (∼ 75%) in some African countries including Nigeria. The official statistics from WHO revealed a scary magnitude of drug counterfeit of 25%. This figure is way higher than global estimates (10%) of reported incidents (http://www.who.int/mediacentre/factsheets/fs275/en/). However, those estimates are not concrete because of the limited number of published researches concerning drug counterfeiting [4]. Frequently, the counterfeit-related incidents appear in newspapers and other online resources rather than the biomedical literature. Additionally, much of the ambiguity around the cases of poor drug quality/counterfeit drugs are due to restriction on the accessibility and reluctance by the pharmaceutical and regulatory authorities to publish. However, counterfeit prevalence studies from the national and international organizations still constitute the authentic source of drug falsification estimates. All categories of drugs are vulnerable to counterfeiting. Kelesidis et al. have summarized the counterfeit prevalence of antimicrobial drugs worldwide [4,5]. Numerous publications reported significant falsification of antimicrobials that could go as high as 50% of the worldwide fake drugs. Developing countries account for the majority of antibiotic falsification (78%), an appallingly high rate which undermines already insufficient public healthcare. According to WHO and the United Nations Office on Drugs and Crime (UNODC), the major sources of those substandard or counterfeit antimicrobial drugs are coming from India, China and Thailand. A review on antibiotic counterfeiting indicated an alarming rate of 44%, 30% and 9% in Asia, Africa, and Europe and North America, respectively [6]. Among the antibiotic counterfeits, ␤-lactam antibiotics account for 50% of the cases followed by quinolones (12%), macrolides and lincosamides (11%), tetracyclines (7%) and others (20%) [6]. Assurance of providing quality medicines is key to public healthcare worldwide [7]. The dosage of drugs prescribed is crucial for the therapy of certain diseases like microbial infections. A sub-potent dose administration of drugs (antibiotics) increases the chance of therapeutic failure through microbial genesis of resistance thereby increasing the burden on the healthcare system [8–10]. Resource constraint developing countries suffer in their healthcare system from a lack of analytical and distribution infrastructure to monitor and regulate the quality of medicines until the end user. In poor nations like those from western Africa and some parts of Asia, infectious diseases are a common health issue and logically demand high proportions of anti-infective medications for treating them. Consequently, those medications are in particular susceptible to drug counterfeiting [6,11–14]. The quality of medicines is ensured through the implementation of quality control (QC) and Good Manufacturing Practices (GMP) [15]. The monographs for quality control of almost all avail-

able drugs are reported in different pharmacopoeias. Most of the methods are based on sophisticated analytical tools, for example gas chromatography (GC) or liquid chromatography (HPLC) hyphenated to different detection systems (UV, mass spectrometry, fluorescence, chemiluminescence etc). Apart from the large capital investment necessary for the acquisition of such techniques, high grade solvents required for those techniques, highly qualified technicians for maintenance, and the necessary infrastructure (air conditioning, humidity control, continuous electricity) to operate those equipments are not always available in the developing world. Since constant quality control of medicines is essential for effective healthcare [16,17], measures needed to tackle drug counterfeiting are a highly critical issue in low and middle income countries. Hence, development of simple, robust, sensitive and economic analytical alternatives that are able to be performed independent of laboratories and skilled staff is urgently needed. Different techniques have already been introduced since the perception of necessity of simple, robust and sensitive tools in the 1980s. Different approaches (such as kit based techniques) have been implemented already, but met with little success. Most recently, capillary electrophoresis (CE) based methods are proposed as an economic and efficient alternative to HPLC to combat drug counterfeiting [18]. In this review, we describe CE as a potential analytical tool for miniaturization followed by a discussion on multiple aspects of improvements achieved in the detection system as well as its application in the determination of antimicrobial medication. 2. CE with conductivity detection CE is a straightforward technique requiring only a piece of capillary and minute amounts of solvent and sample. However, developing a robust CE method is not always easy since it is subject to multifactorial influence. CE is reported to couple different detection techniques and hence, method specific adaptation with respect to the detection unit is necessary for CE method development. UV detection is the common detection technique; however, the bulky size of the UV detector occupies a significant part of the equipment. Additionally, the decay of the UV lamp over time may result in increased replacement cost and may not be amenable for integration with portable and chip-based platforms of CE. The application of amperometry in CE analysis is also promising due to significant detection gain, but limited only to those compounds with redox potentiality [19]. Moreover, the fabrication of the amperometric device is not straightforward for the miniaturization of the CEamperometry system. In contrast, conductometric detection (CD) is amenable to fit in the CE platform. The CD detector employs the measurement of conductance which is basically a bulk physical property of substances. This is the reason why CD is termed as a universal detection system. Unlike some of the previously mentioned detection units, CD structurally uses an electronic device. Different CD configurational strategies have been adopted in its early stages of development. The early CD configuration employed was direct electrode insertion into laser induced microholes on the capillary. A wall-jet configuration exploited the lowered background conductivity achieved through ion-exchange, a phenomenon which is alternatively called “suppressed CD”. Other configurations reported for conventional CD are i) deposition of platinum at the capillary outlet [20] and ii) resistance measurement of liquid trapped by the hydrophilic polymer at the capillary end [21]. 2.1. CE - capacitively coupled contactless conductivity detection A relatively new approach termed, CE - capacitively coupled contactless conductivity detection (C4 D) has been proposed as an

P. Paul et al. / Journal of Pharmaceutical and Biomedical Analysis 158 (2018) 405–415

Fig. 1. A simplified diagram of CE - capacitively coupled contactless conductivity detection (C4 D).

alternative to CE-UV. The strong point of CE-C4 D is its ability to detect both chromophoric and non-chromophoric charged analytes. Though the sensitivity issue with C4 D still persists, inclusion of it in the CE system improves the technique further in terms of instrument size, convenience, capillary integrity, cost and possibility to miniaturization. A lot of work has been done on the CE-C4 D simplification and its portability through miniaturization. Mechanically, the proposed C4 D comprises a couple of annular metal electrodes placed coaxially along the capillary (see Fig. 1). An alternating voltage (AC) is applied to one of the electrodes (actuator electrode) and the current passing through the capillary liquid is captured by the other electrode (pick-up electrode). The resulting signal is picked up by an amplifier and is further processed through AC-DC conversion and data acquisition. Zemann et al. and Fracassi et al. independently introduced the concept of C4 D with the aim to circumvent the demerits of contact conductivity detection: detector polarization, detector air-bubble generation as well as electrode corrosion. The authors have implemented C4 D in the less suited CE mode of operation: MEKC, for non-ionic chemical species as well [22]. Moreover, C4 D has been successfully applied to non-aqueous buffer systems for the detection of less polar organic ions [23]. Simultaneous determination of both cationic and anionic species by dual end injection demonstrates the operational flexibility of CE-C4 D [24]. 2.2. Principle of C4 D The concentration and conductance of the sample and the BGE contribute to the generation of the detector signal. C4 D is an improved variant of the CD platform that has certain advantages over the latter. Like CD, C4 D consists of two metal electrodes. However, the electrode pair is not in contact with the reagent. Eventually, electrophoresis-induced electrode decay is eliminated in C4 D. Because of the contactless character, C4 D avoids the air bubble formation, a problem that affects the current flow through the capillary and eventually the separation efficiency. Assembly of CD into the capillary is difficult whereas C4 D simply involves its placement coaxially along the capillary (see Fig. 1). The C4 D detector distance from the capillary inlet can be adjusted in real-time. Another important aspect of conventional CD is the possibility of interference of the detector voltage with the CE separation voltage. With C4 D, this voltage interference is totally abolished so that the two electronic modules work independently. In C4 D, two electrodes are separated by non-conducting material and give rise to capacitance under electric potential. In fact, the electrodes are connected capacitively through the tubing (called coupling capacitance) followed by the double layer capacitance due to BGE in the interior of the capillary. This is why, it is called capacitively coupled contactless CD (C4 D). Capacitance is an integral part of the C4 D circuitry. Normally, two conductors separated by a dielectric medium comprise a capacitor. The amount of charge contained in a capacitor is directly proportional to the surface area of the conductors (electrodes) and inversely proportional to the distance between them. In CE, we see multiple capacitances contributing to the C4 D signal generation and removal of this interference has a constructive effect on the signal sensitivity. Drawing the equivalent electric circuitry helps in better

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understanding of the capacitance contribution in C4 D. The equivalent circuitry (Fig. 2A) of the detector indicates that not only the measured analyte contributes to the response, but also the geometry of the electrodes and permittivity of the dielectrics (capillary material, BGE, solvent). The circuit diagram is further simplified by successively discarding contribution from the electrical double layer capacitance, Cedlc (Fig. 2B), stray capacitance, Cs (Fig. 2C) and sample capacitance, Cliq (Fig. 2D). The super simplified equivalent circuitry now only involves the coupling capacitance, Ccpl and the impedance, Zliq , from the sample band. Principally, C4 D measures the conductance, which is reciprocal to the impedance of the analyte and BGE. The impedance (Z) is basically the sum of resistance (Rliq ) and the product of capacitance (C) and an imaginary constant, i. Ztotal = R liq +iC

(1)

This total signal relates to the BGE and analyte band. For clear understanding of the concept, it is often stated that the peak recorded by the C4 D detector, is related to the difference (Z) between the impedance of the BGE and the analyte band. Z=Z BGE -Z analyte

(2)

Indeed, the conductance will change depending on the presence of analyte within the detector window (distance between the electrodes) of the C4 D. This change in conductance will translate into the analyte peak. 3. Overview of low cost portable electromigration devices Portable analytical devices are preferred for field analysis due to speedy evaluation of the sample, better cost-effectiveness and avoidance of complications associated with sample storage and transportation. Therefore, portable devices should have small dimensions for easy transportation and be operational independent of mains power (battery-driven). The advantage of small dimension electric devices is the long operational lifetime, thanks to the low current flow hence low power consumption. The concept of portability initiated an era of analytical device miniaturization that offers a host of operational, economic and environmental benefits. C4 D is light, compact and easy to fit along the capillary without creating any detection window on the capillary. Therefore, capillary integrity remains unaffected. Incorporation of C4 D into the CE instrument will not only simplify the existing instrument, but also enable easy miniaturization, thereby facilitating the development of a portable unit for field level drug analysis. Several projects have already been implemented in this regard and applied in some African countries. Several commercial variants of CE-C4 D are also available very recently [25,26]. Therefore, developing easy, rapid and robust methods with no or negligible use of toxic organic solvents for the in-field analysis of drugs (especially anti-infectives) is highly indicated. 3.1. Portable devices to fight counterfeit drugs The prospects of portable devices are high in the context of low and middle income countries in order to deal with widespread drug counterfeiting. The easy operation (suitable for non-expert users) and speedy field deployment in particular, are two attractive features that have drawn significant attention among the scientific ¨ et al. reviewed several developing country oricommunity. Hollein ented methods for counter-acting fraudulent drugs [18]. Minilab® was the first inexpensive device for easy detection of counterfeit medicines, but was shown to have some potential limitations [27]. Spectroscopy-based handheld NIR machines sound promising, but with potential drawbacks of lack of quantitation and performance

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Fig. 2. Equivalent circuitry for the CE - capacitively coupled contactless conductivity detection (C4 D).

inconsistencies for formulations with low API to excipient ratio [28]. This review also highlighted the feasibility of several semiquantitative (TLC, HPTLC) and quantitative methods (HPLC, CE). Though HPLC is the golden standard for analytical performance [29], lack of miniaturization and its significant installation and maintenance cost preclude field level deployment. However, HPTLC shows, with recent automation of sample application and detection, high accuracy and precision of the analytical results [30–32], but is still expensive compared to TLC. 3.2. CE miniaturization Electro-migration based techniques are best suited for the desired miniaturization in terms of portability, low acquisition and maintenance cost. Among such miniaturized devices, CE-based portable devices are particularly attractive due to the fact that there is enormous potential for various designs of fabrication, short start-up time and some of the shortest analysis times. Multiple experimental designs for CE miniaturization have been reported in the literature, which are broadly classified into two categories: i) non-chip based and ii) chip- based. The non-chip based CE is simply the downscaled version of the existing commercial equipment. This means that the design of the instrument includes a sample injection system (electrokinetic injection or pneumatic aspiration), a short capillary with variable dimension, a suitable detection system (for example, LED UV, LIF, electrochemical, mass spectrometry, NMR, C4 D etc.) [33] and of course, two metal electrodes for electrophoretic separation. Various levels of automation, ranging from manual to completely automatic benchtop units, have been implemented by different research groups. In contrast, the chip-based technique is rather straightforward. It revolutionized the concept of CE miniaturization leading to the idea of lab-on-a-chip [34–36]. While retaining most of the advantages of non-chip CE, it has the flexibility of mass production, a process where etching the separation and sample channels and printing the electric circuitry occur in a single step.

An account of all available CE based portable devices was given in a recent review on the improvement of portable electromigrationbased devices [33]. Obviously, the miniaturization of CE has certain implications for the detection, which should remain sensitive given the short injection plug and narrow detection window, especially in the chipbased segment. This is most possibly true for detection techniques such as UV, LIF and mass spectrometry. Hence, electrochemical and conductometric approaches emerged as an alternative to the former detection methods. Both electrochemical and conductometric detection require electrodes while the latter has more flexibility over the former in terms of its universal characteristics. The most popular version of the latter approach is C4 D. It is striking that a lot of studies have been performed using CE-C4 D. The C4 D detector is cheap and requires less space allowing further miniaturization as in the non-chip and chip-based devices. 3.2.1. Non-chip based CE-C4 D Considering the numerous benefits and structural simplicity of CE-C4 D, it is a good candidate for instrumental transformation into a portable device with a goal of remote applicability. While most of the detection modes have a compatibility issue in coupling to miniaturized CE, the C4 D detector is rather convenient and easily implemented in both non-chip and chip-based CE. Since CE-C4 D is entirely based on electronic principles, it allows simple instrumental manipulation for considerable miniaturization including high configurational flexibility as well as ease of construction. With such opportunities, multiple authors ventured into developing portable CE-C4 D with diverse aims in mind. In addition to this, continuous improvement in terms of operational convenience has been materialized on the prototype, thereby incorporating a wide range of mechanical features, such as a CE-C4 D device with complete manual operation to semi-automated as well as fully autonomous operation. Chronological details of prototypes along with further structural and functional modifications are documented in the following sub section.

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Fig. 3. Semi-automated in-house made portable single-channel CE instruments deployed in Vietnam. 1) C4 D; 2) Safety cabinet; 3) Grounded manifold, including valves, pumps, flow cell interface and flow splitter; 4) Flow cell interface accommodating the ground electrode and one end of the capillary; 5) Gas-pressurized container for delivery of the background electrolyte; 6) Fused silica capillary; 7) Electronic board and 220 V AC-to-12 V DC inverter and 8) High voltage cable [39].

Hauser et al. was a pioneer of portable CE-C4 D instruments [37]. The operation of this prototype was exclusively manual. This means that buffer flushing, sample application, voltage application with appropriate voltage ramping was performed by an operator. Mai et al. improved it further, switching manual operation to semi-autonomic [38,39] followed by a fully automated, computer controlled version [40,41]. The prototype included a safety cage, a grounded manifold, a flow cell interface incorporating the grounding electrode and one end of the capillary, a gas-pressurized BGE delivery system and a 220 V AC-to-12 V DC inverter. The cost effectiveness and miniaturization of the device were successfully executed in this prototype thanks to the use of gas-pressurization and miniature stop valves instead of an expensive motor-driven syringe pump for fluidic manipulation. The BGE pressure is adjusted by a valve regulator and is monitored with a small gauge. For the semi-automated set-up (see Fig. 3), sample injection is performed from the high voltage end through a siphoning phenomenon [38,39]. However, the fully automated configuration involves sample loading onto a sample loop by a small pump and subsequently aspirated to a narrow tube leading to the flow cell that accommodates one end of the capillary [40,41]. A fraction of the loaded sample is hydrodynamically injected into the capillary by back pressure for a desired time onto the flow cell. The BGE flushing of the flow cell and manifold prior to the flow cell is simply performed by controlling the outlet flow during the BGE propulsion. The author emphasized on the protection of the high voltage circuit by a plexiglass casing with automatic power disruption facility when the casing door is open. Moreover, the device is amenable to function when connected to the main power supply thereby rendering dual functionality in terms of bench-top use as well as portable format. Recently, several in-house purpose-made CE-C4 D emerged [42]. While commercial bench top CE-C4 D instruments are available requiring a good installation infrastructure, a viable option for modest to resource-constrained facilities is the construction and utilization of in-house-built model devices. Marini et al described the analytical performance of a low-cost CE equipment (ECB2 prototype) for the quantification of counterfeit drugs [43]. This equipment consists of a CE device coupled with a detection system based on fixed wavelength (254 nm) light-

Fig. 4. Capillary electrophoresis budget device (ECB2) [43].

emitting diodes (LEDs). The ECB2 prototype (see Fig. 4) is equipped with two manual samplers for inlet and outlet, and a temperature sensor. Instrument operation as well as data acquisition and analysis are performed by “Chromatos”, a software developed at the HES-SO University, Switzerland. This research group developed and validated a method for quinine, and compared the performance of the device with an Agilent CE machine. A research group from Vietnam has been successful in developing and utilizing a purpose-built CE-C4 D [42]. While most of the instrumental miniaturization happened on the CE part, considerable attention has been put into C4 D. A recent outcome of such efforts is an easily constructed miniaturized C4 D integrating all circuitry in the detection cell with battery operation [44,45]. An alternative compact version of a C4 D cell has been described by do Lago et al. [46]. The automated bench-top CE-C4 D system with sequential injection analysis (SIA) described in [47] is a fully automated unit (see Fig. 5) suitable for unattended operation for several days. In general, commercial CE is relatively expensive and not suitable for in-situ use. Hyphenation of a SIA manifold to an in-house built CE unit is relatively easy. A SIA-CE combination offers operational and economic advantages in terms of programmable BGE and sample volume management, automated sample injection with subsequent voltage triggering and data acquisition. The analytical procedures are all performed hassle-free, thanks to the management of fluidics, which is computer-controlled through a graphical interface. The heart of this unit is a motor-driven syringe pump, a rotary valve and a fluid handling loop in-between them. Fluid is first pumped into the holding loop followed by propulsion through the rotary valve into the flow cell interface. Sample is injected firstly by aspiring it into the flow cell followed by syringe pump mediated hydrodynamic injection into the end of the capillary. Actual separation is performed by applying voltage on the detection end of the capillary while the other end in the flow cell is grounded. Automatic BGE replenishing may be coupled to the unit, but everything is housed inside the safety case.

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Fig. 5. The automated SIA-CE-C4 D system for unattended monitoring operation [47].

Fig. 6. The in-house-made portable dual-channel CE system using two individual BGEs. 1a and 1b) C4 Ds; 2a and 2b) High voltage chambers that contain high voltage cables and electrodes; 3) Grounded manifold, including valves, pumps, flowcell interfaces and flow splitters; 4) Electronic board; 5) Power supply [40].

A single capillary CE allows separation of both cations and anions in a single run. Incorporation of C4 D as a detection unit to the CE has the flexibility to modulate the effective length thereby favoring precise control of selectivity and electrophoresis duration as well. The electronic-only feature of CE-C4 D enables usage of multichannel CE with easy manipulation of the existing devices. Multichannel CE-C4 D offers operational flexibility over the conventional single channel counterpart. It allows CE analysis with different voltage, different buffer sets specific to different charged analytes and hence augments operational flexibility of the existing bench-top configuration [48] and portable format [40,49]. The fluidics of multichannel CE-C4 D are facilitated by pneumatic pumping (i.e., compressed air supported pressurization of BGE reservoir) and two or three-port valves for the adjustment of flow direction. The operational aspect of multichannel CE-C4 D is similar to the existing gas-based single channel CE [50]. In dual channel CE-C4 D (see Fig. 6) that employs only a single BGE, both capillaries share the same electrical ground electrode (encased inside the flow cell) for the electrophoresis, but have an individual C4 D for each capillary. The voltage is applied to the detector end of the capillaries. The safety of this device is ensured by encaging everything in the isolation box with a door opening sensitive switch for power interruption. Each channel of this configuration operates independently as an automated gas based

compact CE device [40]. Moreover, separate optimization of the CE condition for positively and negatively charged species is an interesting feature of such an instrument provided a shared BGE. The dual channel CE with different BGE, in contrast, allows concurrent determination of analytes belonging to different categories in a single run [51]. The most interesting part of this CE set-up is that the same principle can be applied for designing and implementing MCE for high throughput analysis. Greguˇs et al. designed a portable semiautomatic CE-C4 D (see Fig. 7) for the analysis of small volumes of biological samples [25]. It can also be coupled to a number of sampling devices, an interesting feature that opens perspectives towards application in diverse fields. Despite the limitations of siphoning-based injection, this device is claimed to be useful for point-of-care clinical applications. 3.2.2. Chip-based (microfluidic) CE-C4 D While a number of research groups explored the idea of portability-oriented miniaturization of this technique, several have already conducted studies on the micro-level miniaturization (chip-based) of CE-C4 D. Implementation of CE in a microchip platform with diverse applicability is not new. Like commercial CE, microfluidic CE has been shown to be compatible with all kinds of detection techniques [52–56]. However, full integration of these detection systems with a glass or polymer-based microflu-

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Fig. 7. A photograph of the instrument (A) and a schematic of the portable CE-C4 D instrument (B). (BGE) Background electrolyte, (HV) high voltage, (V) valve, (W) waste reservoir, (h) the pressure differential applied, (C4 D) capacitively-coupled contactless conductivity detector, (DAS) data acquisition system, (T) tablet, (CP) control panel, (INJ + G) injection interface and ground electrode, (R + HV) rinse interface and the high voltage electrode, (BAT 1, 2) batteries, (ESB) electronic board [25].

Fig. 8. Block diagram of chip-based CE-C4 D: Electrodes (E1 and E2 ), sample or BGE holder (S1 and S2 ).

idic system has not been implemented so far. The complication and sophistication associated with their fabrication perhaps were the reason behind the incompatibility to full integration. However, the provision of full integration of the electrochemical and conductometry-based detectors [55,57] are the only exceptions. Electrochemical based microfluidic chips are similar to conductometric detection in construction pattern, but have an inherent limitation of narrow applicability (only suitable for compounds with a redox potential). The universal conductometric detection format C4 D conversely, shows the potential of parallel miniaturization of both the CE and the detector with full integration, thanks to the miniaturization potential of C4 D (Fig. 8). The fundamental concept remains unchanged as with conventional CE-C4 D, but the convenience associated with physical modification allows customizability to serve the desired analysis. Most often, glass is used for etching microfluidic channels, but full integration of electrodes is easily carried out on polymer-based chips. Unlike conventional CE-C4 D, the chip-based platform has an integrated injection system where electrokinetic sample loading is performed preferentially.

The sample plug is loaded into the cross-section of the sample and separation channel. It has the merits of requiring very low volume of sample and reagent, fast analysis, parallel measurement and functioning without mains power. With the help of advanced technology, it is now possible to etch a separation and sample-in-or-out channel and simultaneously print out electrodes and associated electric circuitry, enabling mass manufacturing. Therefore, chip-based CE-C4 D not only simplifies the device itself, but also reduces the unit cost significantly. As a whole, it becomes an attractive portable package for quality surveillance of essential drugs in resource-constrained countries. However, it also has potential drawbacks like limited separation efficiency and sensitivity as well as imprecise sample loading compared to the conventional type. The design optimization of the chip-based CE-C4 D showed improvement in reproducible sample injection. Since both ends of the sample and separation channel have electric connection, a proper electric scheme can be applied to obtain reproducible sample loading such as pinched [58], floating [59] and gated [60] sample loading schemes. However, despite efforts to overcome such issues, poor precision and sensitivity still persist and need to be addressed in the future, in order to fully exploit its economic advantage in the context of developing countries. The mainstream application of chip-based CE is in the clinical diagnostic, genomic and proteomic research fields so far. Under the current scenario, the application of chip-based CE-C4 D for the determination of antibiotics is still a nascent concept. Considering the pros and cons of chip-based CE-C4 D, the implementation of CEC4 D based methods for antibiotic determination will necessitate significant attention on the precision and sensitivity issues of chipbased platforms.

4. Overview of CE-C4 D application in antibiotic determination The separation efficiency of CE has driven numerous applications in diverse analytical fields. Multiple reviews on the application of CE in pharmaceutical analysis suggest that a plethora of small drug molecules (including members of different antibiotic

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Table 1 CE-C4 D as applied to the determination of antimicrobial drugs. Family (Antimicrobials)

BGE and CE parameters

Reference

␤-lactam (amoxicillin, ampicillin, Pen V and flucloxacillin) ␤-lactam (Amoxicillin) Fluoroquinolone (Ciprofloxacin) Fluoroquinolone (Ciprofloxacin) Aminoglycosides (Amikacin) Aminoglycosides (Amikacin) Aminoglycosides (Kanamycin) Aminoglycosides (Tobramycin) Macrolides and lincosamides (Azithromycin, clarithromycin and clindamycin) Antifolate (Sulfamethoxazole and trimethoprim)

50 mM TRIS and 50 mM L-histidine without pH adjustment, + 25 kV, hydrodynamic injection (5 s at 3.45 kPa) 10 mM TRIS/TAPS (pH 8.4), + 25 kV, short end hydrodynamic injection (1.0 s at 25 kPa) 1.8 mM oxalic acid and 12 mM triethanolamine (pH 8.5), + 25 kV, hydrodynamic injection (0.5 s at 25 kPa) 10 mM sodium citrate without pH adjustment, + 20 kV, hydrodynamic injection (5 s at 3.45 kPa) 20 mM TRIS and 0.3 mM CTAB with L-histidine, pH adjusted to 6.6, −30 kV, hydrodynamic injection (5 s at 3.45 kPa) 30 mM malic acid and 10 mM 18-Crown-6 with L-arginine, pH adjusted to 4.1, + 30 kV, hydrodynamic injection (1 s at 25 kPa) 40 mM MES, 40 mM L-histidine and 0.6 mM CTAB (pH 6.35), −30 kV, hydrodynamic injection (5 s at 3.45 kPa) 25 mM MES and 0.3 mM CTAB with L-histidine, pH adjusted to 6.4, −30 kV, hydrodynamic injection (5 s at 3.45 kPa) 20 mM MES, 40 mM L-histidine and 0.6 mM CTAB without pH adjustment (6.39), −30 kV, hydrodynamic injection (5 s at 3.45 kPa) Lithium phosphate, pH adjusted to 7.1, + 30 kV, hydrodynamic injection (5.0 s at 25 kPa)

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classes) have been analyzed by CE. The majority of these research works involved optical detection systems (UV, LIF) with several reports on hyphenation to other detectors (amperomety, conductometry, mass spectrometry). While we observe the popularity of the CE-UV and CE-LIF platforms in the commercial domain, other detection choices are predominantly used in academic research. However, CE-C4 D, a relatively new player in the field, has gained significant attention among the research community world-wide since its inception in 1998. Application of the CE-C4 D concept in the analysis of drugs including antibiotics has been extensive. A brief account of all the antibiotic analyses being implemented with this concept (see Table 1) is given in the following section: Three methods for the determination of drug(s) from four classes of antibiotics (penicillins, fluoroquinolone, macrolides and lincosamide) were developed. A quality by design approach (QbD) was implemented in the development of a simple and robust CE-C4 D method for the determination of ciprofloxacin. The drug selection was guided by its popularity as a broad spectrum fluoroquinolone antibacterial drug in the context of developing countries [61]. The method comprises a 10 mM sodium citrate buffer without pH adjustment (pH 3.85), with two minutes of inter-injection BGE rinsing and + 20 kV electrophoresis in a short analysis time (3 min). The obtained results were satisfactory in terms of precision and accuracy. Azithromycin, clarithromycin and clindamycin are three broad spectrum antibiotics with potential risk of counterfeiting in developing countries due to their high price and prescription rate. Commercial formulations [62] of these three antibiotics were determined concomitantly by a rapid (3 min), simple and robust CE-C4 D method. An easy-to-perform strategy was implemented in all methods, viz. minimum number of steps involved in sample and BGE preparation, shortest possible duration of analysis with nonstringent operator skills and low reagent consumption. A BGE of 20 mM 2-N-(morpholino) ethane sulfonic acid (MES) and 40 mM L-histidine without pH adjustment (pH 6.39) was used including 0.6 mM cetytrimethylammonium bromide (CTAB), an EOF modifier. The user-friendly method ensures an easy operation by implementing a weigh-and-dissolve approach, a concept that is likely to eliminate the necessity of skilled operators. The major areas that required optimization with respect to the analytes, were the BGE composition and C4 D parameter settings with respect to the BGE. The electrophoresis was performed at −30 kV for 6 min with 3 min of inter-injection BGE rinsing. The method performance was satisfactory in terms of sensitivity, precision and accuracy. More-

[69] [68] [61] [64] [67] [66] [65] [62] [70]

over, it was demonstrated that the robustness of the method is appropriate. Recently, the determination of four penicillin antibiotics was developed [63]. This paper emphasized on the detection of substandard and counterfeit WHO-recommended penicillin formulations for use in developing countries. The operator-friendly method performed electrophoresis at 25 kV in a 50 mM TRIS and 50 mM L-histidine solution. The obtained data showed reasonable precision and accuracy for the determination of the four ␤-lactam antibiotics amoxicillin, ampicillin, phenoxymethyl penicillin and flucloxacillin. The other instrumental parameters remained the same as for ciprofloxacin. El-Attug et al. demonstrated the strength of CE-C4 D for the determination of a very polar aminoglycoside with no chromophoric activity like amikacin and its related substances [64]. The BGE chosen for optimum resolution of the analyte and its related substances included 20 mM MES and 0.3 mM CTAB, with pH adjusted to 6.6 by L-histidine. The optimized instrumental parameters enabled baseline separation of amikacin and its reported related substances within 6 min of analysis. For the analysis of tobramycin and related substances, El-Attug et al. used 25 mM MES, pH adjusted to 6.4 with L-histidine and 0.3 mM CTAB as BGE for optimal separation among the analyte and its potential impurities [65]. The same author applied CE-C4 D for the determination of kanamycin and its related substances [66]. Being a non-UV active molecule, kanamycin is also a good candidate for determination with this technique. The author employed a slightly different BGE containing 40 mM MES and 40 mM L-histidine along with 0.6 mM CTAB as EOF modifier (pH 6.35). El-Attug et al. authored a CE-C4 D method for the simultaneous determination of amikacin and urea in the bronchial epithelial fluids of neonates [67]. This BGE included 30 mM malic acid and 10 mM 18-Crown-6 with pH adjusted to 4.1 using L-arginine. The optimized method showed good precision, accuracy and linear concentration range (0.6–24 mg L−1 for urea). Montes et al. described a CE-C4 D method for the determination of ciprofloxacin in milk and pharmaceutical samples [68]. The author compared the CE-C4 D method performance with batch injection analysis coupled to amperometric detection (BIA-AMP) and HPLC with UV detection. A 1.8 mM oxalic acid and 12 mM triethanolamine BGE (pH 8.5) showed good results in CE-C4 D with satisfactory sensitivity, recovery, high linear range, and precision compared to the BIA – AMP method. A 25 kV normal polarity elec-

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trophoresis was significantly faster than HPLC with comparable results. The precision of the method was improved by the use of the internal standard (IS) lithium which is a normal practice in CE. Another CE-C4 D based method for rapid, concomitant determination of amoxicillin, clavulanate and potassium was reported by Marra et al. [69]. The authors used a BGE of 10 mM tris(hydroxymethyl)aminomethane/ tris(methylamino)propanesulfonic acid (TRIS/TAPS) with pH adjusted to 8.4. Less than a minute of electrophoresis at 25 kV following short end injection rendered an electropherogram with all three analytes in the order of potassium, amoxicillin and clavulanate. Given such a small analysis time, the method was 40-fold faster compared to conventional HPLC, making it more economic for the routine determination of pharmaceutical formulations. A combination of trimethoprim and sulfamethoxazole, a very effective antimicrobial formulation, has successfully been determined by da Silva et al. with a fast CE-C4 D method [70]. They used an inorganic lithium phosphate salt in the BGE, adjusted to pH 7.1. Interestingly, two C4 D detectors were positioned coaxially at two different effective lengths (10 and 39 cm) in order to achieve better resolution between the system peak (EOF) and trimethoprim. They reported a high frequency of analysis (75 h−1 ) with the first detector, in addition to other aspects of the method, such as satisfactory precision, accuracy and good selectivity for the selected formulations within a very short duration of analysis. Quek et al. demonstrated the potential of CE-C4 D for biomedical and environmental application [71]. These authors applied the technique for the determination of 13 pharmaceutical products, which are being considered as emerging pollutants, in water samples. The pharmaceuticals included in this work range from NSAIDs, antibiotics, antihistaminic and anticonvulsant compounds, lipid regulators and beta-blockers. The BGE comprised 9 mM TRIS and 5 mM lactic acid with pH adjusted to 8.0. However, the optimal resolution among the analytes was achieved through a combination of three cyclodextrins (CyD) (0.025% ␥-CyD, 0.075% hydroxyl-␤-CyD and 0.15% dimethyl-␤-CyD) with 5% n-propanol as organic modifier. Sample injection was performed by siphoning (10 cm height difference for 10 s). Acceptable precision was obtained through 1 min of successive water and BGE flush in-between injections followed by normal polarity electrophoresis at + 15 kV applied voltage. In a nutshell, it can be observed that the application of CE-C4 D in the determination of antibiotics is increasing. Short analysis time, small volume of sample and reagent and portability aspects will enable it to be adopted in the developing countries for counterfeit antibiotic monitoring.

5. Concluding remarks Given the simplicity of CE-C4 D, the application of this technique to the analysis of antibiotics is promising. Simple and economic application are the two characteristics of CE-C4 D that make it a promising analytical tool to deal with the threat of counterfeit antibiotics in the developing world. Implementation of portability on the benchtop version of CE-C4 D allowed emergence of inexpensive devices. Additionally, significant understanding and improvement have already been achieved in terms of electronics and sensitivity of CE-C4 D. Such information enabled further miniaturization of CE-C4 D into microchip CE-C4 D with significantly reduced cost. This miniaturized platform not only adds to the portability aspect but is also consistent with the economy, sample and reagent requirements, energy consumption and off-site application. However, most of the non-chip and chip-based devices are prototypes and further attempts are needed so that low income nations can benefit from their analytical and economic advantages. The number of studies involving portable CE-C4 D in the analysis of

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antibiotics is still increasing; therefore, it is necessary to assess the compatibility of already developed methods in both platforms of CE-C4 D.

6. Future prospects Given the potential of CE-C4 D in miniaturization and separation efficiency, we expect it to be adopted for the routine quality check of drugs’ compliance to the compendial requirements. Besides, resource-constrained countries can capitalize on the economic benefits of the system as a whole. As very simple and robust CEC4 D based methodologies for the antibiotics are emerging, it is now high-time to utilize inexpensive CE-C4 D platforms, via partner collaboration, for quality assessment of antibiotic formulations. In this regard, the inter-laboratory testing of newly developed methodologies is a supporting step, which is suggested for the next phase. An inter-laboratory testing of the methodologies developed by our research group can be a starting point. In doing so, the compatibilities of these methods with different CE-C4 D platforms will be established. Eventually, this will enhance the smooth access of poor populations to good quality anti-infective drugs. Additionally, such initiatives will help, in a significant way, to restore faith in the public healthcare institutions in the developing countries. Such endeavor will not only benefit the poor segment of the world community for safer entrance to medication, it will also lessen the toxic burden of organic chemicals, a by-product of conventional analytical tools, on nature.

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