Ion chromatography coupled with fluorescence/UV detector: A comprehensive review of its applications in pesticides and pharmaceutical drug analysis

1 Introduction

The dramatic rise in the consumption of pesticides, pharmaceutical drug, and their derivatives during the last few decades has aroused serious concern about the potential adverse effects on the ecosystem and human health. Their continuous disposal in the environment is the cause of their incorporation into all kinds of matrices such as crops, vegetables, fruits, soil, water, serum, urine which has ultimately appeared as a threat to human health and ecosystem (Białk-Bielińska, et al., 2016, Olsson, et al., 2004, Tao and Carta, 2008).

United States Environmental Protection Agency (US EPA) defined pesticides as “the substance used to repel, control or kill insects, weeds, rodents, bacteria, fungi or any other organisms and regarding their mode of action, named insecticides, herbicides, rodenticides, bactericides and fungicides. The classification of pesticides based on the target organism is shown in Fig. 1 (a). According to the 2015 EU pesticides database, 500 pesticides out of 1300 active substances were approved for utilization in European states. According to the US EPA estimate, approximately 56 billion dollar pesticides were consumed throughout the world in 2012 and is increasing each year (EU, 2015, US EPA, 2015).

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Fig. 1 General classification of (a) pesticides based on target organism and (b) pharmaceutical drugs based on mode of action.

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Similarly, pharmaceutical drugs and antibiotics are pseudo-persistent as a result of their continuous mixing into the ecosystem (drinking, ground, and surface water) mainly through human excretion and later partial elimination of pharmaceutical compounds in the wastewater treatment plants (WWTPs) (Vazquez-Roig, et al., 2012, Vazquez-Roig, et al., 2013). There is a continuous rise in annual drug consumption and in 2010 the pharmaceutical drug market reached US$ 875 bn. Several potential risk assessments on the aquatic environment were carried out and according to WHO, the appearance of bacterial resistance against pharmaceutical drugs is regarded as a serious threat to human health. Therefore, all drugs are classified based on their mode of action or therapeutic effect as shown in Fig. 1(b) and WHO has listed all important pharmaceuticals (WHO, 2014, ESRIC, 2010).

Research on their further toxic effect is under investigation. Some notable toxic effects imparted by pharmaceutical drugs include hair loss, rashes, allergies, nausea, vomiting, severe gastrointestinal lesions, constipation, diarrhea, drowsiness, ulcers, kidney failure, hypersensitivity, congenital malformations, infertility, and adverse reproductive outcomes (Broto, et al., 2017). Similarly, pesticide toxicity, stability, and ability to bioaccumulate, impart a more deleterious effect on human beings and the environment (Fenik, et al., 2011). Hence, both pesticides and pharmaceutical drugs (including antibiotics) through a systematic chain are exposing to human beings and causing adverse effects as layout shown in Fig. 2(a & b).

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Fig. 2 (a & b) Systematic cycle of pesticides and pharmaceutical drugs.

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Therefore, to avoid potential health risks to humans from continuous exposure to pesticides and pharmaceutical drugs, increasing efforts are made to monitor their presence and concentration in different matrices of life. So far, many analytical methods have been developed by hyphenation of reversed phase separation technique with UV or mass spectrometric detector for analysis of pesticides and pharmaceutical drugs, and for convenience, these methods have been reviewed (Baciu, et al., 2015, Białk-Bielińska, et al., 2016, Bottoni and Caroli, 2018, Esteve-Romero, et al., 2016, Fenik, et al., 2011, Gouda, et al., 2013, Hernández, et al., 2014, Hernández, et al., 2008, Iguiniz and Heinisch, 2017, Martínez-del-Río, et al., 2013, Mohamed, et al., 2016, Siddiqui, et al., 2017, Tadeo, et al., 2004). The gas chromatography (GC) and high performance liquid chromatography (HPLC) coupled to mass spectrometry (MS) and diode array detector (DAD) is the most frequently used technique for their separation and analysis. The selection of separation mode potentially depends on the nature of pesticides /pharmaceutical drugs (i.e. vapor pressure). For instance, the low vapor pressure and thermal lability of polar pesticides /polar drugs make LC-MS/DAD more amenable over GC–MS, which needs complex derivatization for their separation (Barron and Gilchrist, 2014). Recently, there is growing interest and demand in analytical chemistry regarding the development of hyphenated liquid chromatography that helps to reduce cost, analysis time, multiple analyte analysis in a single run, minimum consumption of organic solvent, information of analyte occurrence, automation, improved analytical performances (sensitivity, selectivity, matrix-free, accuracy, and precision) (Gouda, et al., 2013, Hernández, et al., 2014). LC‐MS appeared a better alternative to GC–MS for their analysis due to its high sensitivity and no need for derivatization of non‐volatile analytes (otherwise required by GC–MS). But its substantial drawbacks are (i) if analytes poorly chromatographically separated then phenomenon of co-suppression could occur, where the signal of analytes of interest can be lost at the mass spectrometer interface if those particular analytes have low ionization efficiencies; (ii) inability to simultaneously analyze multi-class analytes; (iii) high cost for routine analyses and (iv) need of experienced workers. These factors have limited its use and access in developing countries (Hernández, et al., 2008, Jin, et al., 2012, Muhammad et al., 2018a, 2018b, 2018c, 2018d).

Despite the growing use of hyphenated reverse phase chromatography, the need of an organic solvent in high content, cost of mass spectrometric instrumentation, limited pH range compatibility normally less than 7 of reverse phase C18 column, inability to separate structurally similar and polar analytes and limited column durability drove analysts toward alternative techniques i.e., ion chromatography (Muhammad et al., 2018a, 2018b, 2018c, 2018d).

2 Ion chromatography

In 1975 Small et al., introduced ion chromatography (IC) for the direct, rapid, and sensitive determination of inorganic ions (anions, cations) by coupling conductivity detector to a suppressor ion-exchange column (Small, et al., 1975). Soon in 1979 Gjerde et al., upgraded its use by developing a simple and cost-effective non-suppressed IC technique by running low conductivity eluent. This concept was successively spread further for the separation of ionic, polar and ionizable organic analytes along with inorganic ions (Gjerde, et al., 1979) and led to the introduction of hyphenated ion chromatographic technique, in which it is coupled with conductivity, UV–Vis absorbance, amperometric, potentiometric, light-emitting (atomic emission spectroscopic, atomic fluorescence, molecular fluorescence, and luminescence spectroscopic detection) and MS detector (Buchberger, 2001). The IC braced to non-specific detectors such as conductivity, ultraviolet, electrochemical, fluorescence, and mass spectrometry, has been widely used for over 35 years for the determination of inorganic and organic analytes in simple and complex matrices. Several reviews about IC coupled with conductivity detectors are available in the literature (Fritz, 2000, López-Ruiz, 2000, Paull and Barron, 2004, Shaw and Haddad, 2004). For, instance, Dicinoski et al., reviewed the applications of hyphenated IC for the detection of explosive residues in 2006 (Buchberger, 2001, Dicinoski, et al., 2006). Similarly, L. Barronet et al., reviewed the application of IC coupled with MS for the quantitative determination of polar and ionic species in environmental and forensic explosives (Barron and Gilchrist, 2014). Now it is appeared a better alternative to reverse phase chromatography in the pharmaceutical and drug analysis (Bhattacharyya, 2012, Ravichandran, 2012, Pohl, et al., 2012, Jenke, 2011).

However, no review paper has been reported so far pertaining to the application of advantageous hyphenated ion chromatography-ultraviolet/fluorescence (IC-UV/FLD) technique for the determination of pesticides and pharmaceutical drugs in various matrices, despite growing reports in past decades, as shown in Fig. 3 (a). Therefore, the entire literature from 1980 to 2020 was retrieved via Scopus, Web of Sciences, Google Scholar, Scifinder, Wiley, and ScienceDirect by inserting the terms, “ion chromatography”, “pharmaceutical analysis” and “pesticide analysis” as keywords, separately.

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Fig. 3 (a) Hyphenated IC-FLD/UV based publication per year IC; (b) hyphenation of a non-suppressed ion chromatograph to fluorescence detector (FLD) and (c) hyphenation of a non-suppressed ion chromatograph to post-column reactor and fluorescence detector (IC-hv-FLD) (Subhani, et al., 2013).

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The aim of this review is as follows:

Firstly, this review provides a comprehensive overview of the modified and innovative hyphenated IC-FLD/UV methodology that has been used until now for the analysis of pesticides and pharmaceutical drugs in various matrices. Both classes of analytes are separately elaborated and their respective comprehensive discussions and up-to-date tables are summarized in aiming to provide a detailed discussion related to the new trends in their (pesticides and pharmaceutical drugs) determination by utilizing IC-FLD/UV technique. Secondly, comparison, significance, and advantages of this cost-effective technique over the previously used technique are highlighted. Thirdly, the evolution of this technique and challenges associated with its direct application to complex samples (biological, food environmental) are elaborated. Fourthly, future perspectives and outlooks were outlined to further improve the hyphenated IC techniques in various ways to expand its applications for the determination of organic analytes from widespread samples.

3 Application of hyphenated IC-FLD/UV technique for the determination of pesticides in various matrices

The fabrication of a simple non‐suppressed IC‐UV/FLD or ion chromatography hyphenated to online photochemical derivatization-fluorescence detector system (IC-hv-FLD) is comprised of a pump (IC or HPLC), an injection valve, an ion exchange analytical column (depends upon nature of analytes) fitted in a thermostat, a post‐column photochemical reactor if analytes are non-fluorescent, and a fluorescence or UV detector as shown in Fig. 3 (b & c).

S. Simon et al., introduced a simple and cost-effective method based on online UV photo-oxidation (decomposition) for the determination of four organoarsenic analytes including arsenocholine (AsC), arsenobetaine (AsB), tetramethylarsonium ion (TMAs+) and trimethylarsine oxide (TMAO) by coupling ion chromatography (cation exchange) with atomic fluorescence spectrometry with hydride generation (HG–AFS). IC–UV–HG–AFS appeared a better alternative to replace ammonium ion or pyridine based mobile phase with sodium hydroxide, nitric acid, and potassium nitrate solution. IC compatible mobile phase (NaOH, KOH, KNO₃) enhances the fluorescent signal and IC developed method separates and analyzes four analytes in oysters and mussels within 20 min including 10 min of re-equilibrating the analytical column prior to subsequent injection. Hence, this method with excellent sensitivity (4 – 12 µg/g), reproducibility (less than 3%), and selectivity for the analysis of trace organoarsenic species in marine samples was proved an effective and attractive alternative to liquid chromatography-Inductively coupled plasma mass spectrometry (LC–ICP/MS) having limit of detection in the range of 16 – 30 µg/g with minimum cost (Simon, et al., 2004).

Later on, Qamar Subhani et al., introduced a simple, green (in terms of low content of organic solvent), sensitive and selective ion chromatographic method for the simultaneous determination of two pesticides including imidacloprid and via direct online photochemical derivatization fluorescence detection (IC-hv-FD) in basic media without using the extra pump as shown in Fig. 3(c). IonPac® AS11 anion-exchange column with 40 mM KOH and only 10% (v/v) acetonitrile (ACN) was used for their isocratic separation in less than 10 min. Because, at these optimum condition both analytes (imidacloprid and carbendazim) ion-exchange and adsorptive interactions with IonPac® AS11 column, that has highly cross-linked substrate material EVB/DVB polymer functionalized by alkanol quaternary ammonium functional groups were overcome for their efficient and clean separation with good peak shape and resolution. Effect of various parameters including UV irradiation, pH, and nature of basic media was observed to have the maximum Relative Fluorescence Intensity (RFI) of pesticides in alkaline medium. The developed method was successfully applied for the determination of imidacloprid and carbendazim in real water (ground, lake, and river) with an excellent limit of detection (7.8 and 68 µg L^-1) and recoveries (90–102%) as shown in Fig. 4(a) (Subhani, et al., 2013). This study was further extended for the sensitive photo-induced fluorescence determination of thiacloprid neonicotinoid pesticide in two real water samples (groundwater, lake water) as shown in Fig. 4 (b). The optimized conditions involve the use of 10 mmol/L NaOH as a mobile phase having little content of ACN organic solvent (10% (v/v)) for the isocratic ion chromatographic separation of thiacloprid on an Ion Pac® AS 11 (250 mm × 4 mm i.d; 13 μm p.s) column. The developed method has shown excellent analytical figures of merit such as wide linear range (0.04 – 10.0 mg/L), excellent LOD (9.9 μg/L), and satisfactory recoveries (95.5% − 114.0%) with RSD less than 1.7% for septet measurement. This method appeared simple, cost-effective, selective, and sensitive than previously developed photometric methods (Subhani, et al., 2014). By understanding the successful application of this hyphenated system (IC-UV-FLD), this technique was further utilized for the analysis of other non-fluorescent neonicotinoid nitenpyram and its metabolite 6-chloronicotinic acid in environmental samples (soil and lake water samples). The extraction of both analytes was carried out by applying µ-QuEChERS method in order to reduce the consumption of chemical and organic solvents. Both analytes were simultaneously separated on the IonPac™ AS11-HC column proceeded by the IonPac™ AG11A guard column by pumping 30 mM NaOH having 10% ACN as a mobile phase as shown in Fig. 4(c). This complete separation based optimized method allowed good linear response (r > 0.999), excellent LOD (0.101–0.132 µg/L) with precision ≤ 6.50%. This approached appears sensitive, selective with zero, or minimum matrix effect (Muhammad et al., 2018a, 2018b, 2018c, 2018d). Similarly, other neonicotinoid insecticides namely acetamiprid and its main metabolite 6-chloronicotinic acid, were also analysed by this simple and cost-effective technique (IC-hv-FLD), in environmental samples (groundwater, irrigation water). Both non-fluorescent neonicotinoid acetamiprid and its metabolite 6-chloronicotinic acid exhibited optimum photoinduced fluorescence intensity (PIF) at λ_ex/λ_em = 257/382 nm and λ_ex/λ_em = 231/370 nm, respectively. After an appropriate sample pretreatment, both analytes were simultaneously separated on the IonPac™ AS11-HC column preceeded by the IonPac™ AG11A guard column by pumping 40 mM NaOH mobile phase having very low content of acetonitrile (5%, v/v). The developed method exhibited good linearity for both analytes in the range of 0.050–10 μg/mL with correlation coefficient>0.9993, low limit of detection (0.025–0.0072 μg/mL), satisfactory recoveries (98.02–116.00%) with high inter- and intraday precision (RSD ≤ 3.02%) (Subhani, et al., 2020).

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Fig. 4 (a) IC-hv-FD chromatograms of three real water samples spiked level 0.5 mg/L using IonPacs AG11 (50 mm × 4 mm i.d; 13 mmp.s) proceeded by IonPacs AS11 (250 mm × 4 mm i.d; 13 mm p.s and with 40 mM KOH having 10% (v/v) ACN as a mobile phase. Fluorescence detection at λ_ex/λ_em = 332 nm/367 nm for Imidacloprid while λ_ex/λ_em = 247 nm/470 nm for carbendazim.(1) Blank ground water; (2) spiked ground water, (3) blank canal water; (4) spiked canal water; (5) blank river water; (6) spiked river water samples, (b) Chromatograms of real water samples blanked and spiked at 2.0 mg/L (1) blank of ground water (2) spiked ground water (3) blank of lake water (4) spiked lake water, (c) IC-hv-FD chromatograms of four samples lake water; soil; rain water and sewage water samples using IonPac™ AS11-HC (250 mm length × 4 mm i.d; 13 μm p.s) column and 30 mM NaOH having 10% ACN as mobile phase. The fluorescence detection at λ_ex/λ_em = 236/356 nm for nitenpyram (NTP) and λ_ex/λ_em = 265/352 nm for 6-chloronicotinic acid (6-CNA) (Subhani, et al., 2013, Subhani, et al., 2014, Muhammad, et al., 2018).

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Realizing the need and demand of this cost-effective, sensitive, and green (in terms of low solvent consumption) hyphenated IC system, its application was further extended for the determination of neonicotinoids in complex food samples. The complex nature and composition of food samples forced to modify the previously used IC-UV-FLD technique and µ-QuEChERS sample preparation. This system was modified into two dimensional IC system (2D-IC) for the determination of two non-fluorescent neonicotinoids (clothianidin and imidacloprid) in six complex food samples such as apple, ginger, honey, durian, tomato, and cucumber. Sample preparation and extraction of target analytes from selected samples were achieved by the modified and miniaturized µ-QuEChERS method. Afterward, the matrix interferences from the sample extract were eliminated by utilizing an anion exchange column (IonPac® AS11-HC) in the first dimension of this 2D-IC system. After the elimination of matrix interferences, the target analytes of interest (imidacloprid and clothianidin) were isocratically separated on IonPac® AS12A column (250 mm × 4 mm i.d; 13 mm p.s) preceded by an IonPac® AG12A guard column (50 mm × 4 mm i.d; 13 mm p.s) and determined by passing through a homemade photoinduced fluorescent chamber before their sensitive fluorescence determination. The developed method exhibited excellent analytical figures of merit LOD (0.035–0.154 mg kg⁻¹), inter and intra-day precision (0.12–12.36%), and with no or minimum matrix effect (Muhammad et al., 2018a, 2018b, 2018c, 2018d).

Apart from the fluorescence activation, the UV photolysis pretreatment technique was also utilized for the degradation of N-nitrosodiethylamine (NDEA) into NO₂ ^_ and NO₃ ^_, which was subsequently determined by ion chromatography coupled with a UV detector. It was observed after irradiation of 10–20 min maximum NDEA photodegraded into NO₂ ^_ and NO₃ ^_, while NO₃ ^_ concentration increased after 20 min irradiation. Apart from this, other factors like pH had a direct effect on the formation and oxidation of NO₂ ^_ into NO₃ ^_. This appeared a feasible and accurate method for the determination of nitrosamines from the food products as photoproduced NO₂ ^_ and NO₃ ^_ concentrations appeared equivalent to the initial NDEA prior to the photolysis (Li, et al., 2016).

N. Muhammad et al., developed a simple, selective and immensely sensitive isocratic method for the simultaneous determination of two plant growth regulators (PGRs): 2-(Naphthalen-1-yl)acetamide (NAD) and 2-(1-Naphthyl)acetic acid (NAA) from ten food samples. Both PGRs from these samples were extracted by an appropriate sample preparation technique namely QuEChERS method, and their clean separation and sensitive determination was performed by using ion chromatography braced with fluorescence or ultraviolet detector (IC-FLD/UV). The developed methods exhibited good linearity (r² ≥ 0.980), satisfactory recoveries (76.3–112%) with a limit of detection and limit of quantification as low as MS (0.08–6.6 ng/kg and 2.2–20 ng/kg) in basic media (Muhammad et al., 2018a, 2018b, 2018c, 2018d).

Y. Miyake et al., implemented a combustion ion chromatography (CIC) method for determination of semi- and non-volatile organic halogens (SNVOXs), polychlorinated dibenzofurans (PCDFs), polychlorinated dibenzo-dioxins (PCDDs), and co-planar polychlorinated biphenyls (Co-PCBs) in fly ash and flue gas samples at trace level with excellent recoveries (97–105%) with RSD less than 4% (Miyake, et al., 2007). Table 1 shows a detailed application of hyphenated IC for the determination of a number of pesticides in various matrices.

4 Application of hyphenated IC-FLD/UV technique for determination of pharmaceutical drugs in various matrices

X. Ding et al., first time successfully used high-performance ion chromatography-UV (HPIC-UV) to analyze tetracycline (TCs) antibiotics including oxytetracycline (OTC), chlortetracycline (CTC), tetracycline (TC), 4-epitetracycline (ETC), anhydrotetracycline (ATC) doxycycline (DC) and 4-epianhydrotetracycline hydrochloride (EATC) in the real milk sample. Analytes from target samples extracted by vortex mixing with disodium salt of ethylenediaminetetraacetic acid (Na₂EDTA)–McIlvaine buffer solution (pH 4.0) and followed by filtration of the obtained mixture through a 30 000 Da filter paper or membrane. Afterward, all TCs were isocratilcally separated by utilizing a Dionex OmniPac PCX-100 a polymeric cation-exchange analytical column (250 × 4 mm I.D.) with 0.2 mol/L hydrochloric acid–27.9% acetonitrile within 12.0 min as structure and chromatogram are shown in Fig. 5(a). The developed method showed good selectivity, reproducibility, precision, and detection limit (0–20 µg/L) with a minimum consumption of organic solvent content. Hence, less hydrophobic HPIC appeared more advantageous compared to HPLC (Ding and Mou, 2000). After a successful analysis of these high molecular weight antibiotics, a similar ion chromatographic method was developed for sensitive fluorescence determination of five 4-hydroxycoumarin rodenticides namely warfarin, bromadiolone, flocoumafen, coumatetralyl, and brodifacoum in animal liver tissues. After ethyl acetate-based, solid-phase extraction of rodenticides from liver tissues, their clean gradient IC separation was perforedon an IonPac® AS11 analytical column by using only 10% acetonitrile as an organic modifier. The sensitive fluorescence detection was carried out at λ_ex /λ_{em =} 270 nm / 380 nm as structure and separation chromatogram is shown in Fig. 5(b). The developed method showed better average recoveries in the range (81–98%), the excellent limit of detection in the range 0.004–0.010 mg /kg with inter-day and intra-day-to-day precisions (RSD) were less than 8.5% for all five rodenticides in real animal liver tissues sample. Hence, developed IC-FL method appeared sensitive, selective, accurate, green (in terms of low content of organic solvent) for the determination of five 4-hydroxycoumarin rodenticides in complex animal liver tissues (Jin, et al., 2007).

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Fig. 5 (a) Structure and chromatogram of four tetracycline antibiotics (1) oxytetracycline (OTC); (2) tetracycline (TC); (3) chlortetracycline (CTC); and (4) doxycycline (DC) standard solution by using OmniPac PCX-100 analytical column (250 × 4 mm I.D.) with mobile phase 0.2 mol/L HCl + 27.9% (V/V) CH3CN, (b) structure and chromatogram of five rodenticides (1) warfarin; (2) coumatetralyl; (3) bromadiolone; (4) flocoumafen; (5) brodifacoum obtained by using IonPac® AS11 analytical column (250 mm × 4.0 mm, Dionex) protected by IonPac® AG11 guard column (50 mm × 4.0 mm, Dionex) with KOH/10% MeCN mobile phase in gradient mode (0 → 2.0 min, 2.0 mmol/L KOH; 2.0 → 7.0 min, 2.0 → 20.0 mmol/L KOH in linear gradient mode, and 7.0 → 10.0 min, 20.0 mmol/L KOH at flow rate of 1.0 mL/min (Ding and Mou, 2000, Jin, et al., 2007).

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A rapid and efficient ion chromatographic-indirect UV detection method was developed to avoid the need for complex derivatization for the determination of four bisphosphonates including etidronate, pamidronate, clodronate, and alendronate in pharmaceuticals or bulk material. A Phenomenex SphereClone SAX (250 × 2.0-mm, 5 μm) anion exchange column was used for their separation by running 20 mM sodium citrate buffer (pH 4.6) at a flow rate of 0.25 mL/min and sensitive detection was carried out at 222 nm. It was investigated that pH has a significant effect on the simultaneous separation of three bisphosphonates. Therefore, the optimized concentration of sodium citrate buffer allowed good sensitivity due to the wide difference in analyte and eluent absorbance. Besides, good resolution among system, water, and analyte peak was observed. Hence, the developed method showed good analytical figure of merit linearity (50 to 500 μg/mL), LOD (50–100 μg/mL), and recoveries (97.12–102.92%) with precision 2% (Fernandes et al., 2007a, 2007b).

Philip Zakaria et al., coupled suppressed ion chromatograph to UV detector to demonstrate the isocratic, gradient or multi-step eluent profiles separation of twenty-seven acidic pharmaceutical drugs including (Phenol, valproic acid, beta naphthol, tropic acid, 1-naphthoic acid, acetylsalicylic acid, 5-Methyl-5-phenylhydantoin, Ibuprofen, captopril, naproxen, mefenamic acid, ketoprofen, fenbufen, sulindac, tolfenamic acid, flufenamic acid, indoprofen, chlorothiazide, diclofenac, chloramphenicol, furosemide, indomethacin, cortisone, prednisolone, hydrocortisone, trichlormethiazide, althiazide). The retention model of inorganic ions which solely designed based on electrostatic interactions between the stationary phase and ions was applied for the separation of pharmaceutical organic ions and encouraging results were obtained as little discrepancy was observed between modeled and observed retention time despite the presence of a varying degree of hydrophobic interactions. Therefore, various types of instrumental configurations were designed and investigated to overcome obstacles associated with the use of organic solvents in the mobile phase, due to their incompatibility with the suppressor and electrolytically derived hardware and eluent. The feasible configuration permitted the organic modifier containing stream, to bypass the non-compatible parts such as eluent generator, before entering the injection valve and separation column. In addition, this study provides a future outlook to investigate the application of this or similar system on cationic pharmaceutical compounds (Zakaria, et al., 2009). The ion chromatography also appeared as a useful tool for the characterization of therapeutic proteins and used as a cost-effective technique for quantitative and qualitative evaluation of charge heterogeneity (Fekete, et al., 2015, Lucy, 2003).

Yinying Tao et al., grafted the dextran polymers to agarose beads to investigate the adsorption kinetics and binding capacity of monoclonal antibody (mAb), its experimental cation exchangers capacity was compared with a couple of commercially available with and without dextran grafted agarose-based cation exchangers. It was observed the charged dextran polymers enhanced the adsorption kinetics despite the sudden decrease of pore size, whereas neutral dextran polymers substantially reduced the binding capacities. It was also observed by reducing the protein concentration the effective pore diffusivities substantially increased for charged dextran grafted resins, but uncharged dextran grafted resins had no or minimum dependence on protein concentration (Tao and Carta, 2008). A robust and simple pH gradient-based ion-exchange chromatographic method was developed for the separation and evaluation of charge heterogeneity of monoclonal antibodies. The mobile phase composition involved 6.0 mM piperazine, 11.0 mM imidazole, and 9.6 mM Tris base with different pH values of either pH 6.0 (mobile phase A) and pH 9.5 (mobile phase B) was used to retrieve good analytical figure of merit (Rea, et al., 2011). The same study was further extended for three biopharmaceuticals by employing single component buffer systems as eluents through cation-exchange (CEX) monolithic columns for separation of monoclonal antibody (mAb) charge variants followed by MS detection (Talebi, et al., 2013). Further later, this system was modified by equipping chromatography system with an electrolytic eluent generator (EG) and isocratic pumps to replace external pH gradient delivery with a semi-automated conventional gradient system, in which pre-adjusted pH buffer bottles were mixed with the help of gradient pump. The high purity base or acid was generated by EG for an online proportion of buffer using deionized water as a carrier. Online buffer preparation involved online titration of low molecular weight amines with electrolytically generated acid or base into a static mixing tee to get the required pH. This online approach offered high throughput in terms of labor, mobile phase preparation, and highly reproducible pH profile, which was confirmed by running pH gradients in the range of 8.2 to 10.9 into a polymeric monolith cation-exchange column for high chromatographic resolution and separation of basic therapeutic monoclonal antibodies (Talebi, et al., 2014).

Hong Wei Wu et al., developed an eco-friendly analytical method for the determination of three catecholamines viz. dopamine (DA), epinephrine (EP) and norepinephrine (NE) in human urine by using ion chromatography (IC) followed by chemiluminescence (CL) detection. The CL detection procedure involved the reaction of catecholamines with acidic potassium permanganate and formaldehyde to enhance CL signals. The three catecholamines from urine samples were extracted by vortex mixing of 1000 μL of the sample with 1000 μL acetonitrile. After centrifugation, the supernatant was evaporated/reconstituted before injection into the IC-CL system for their separation on Dionex CS12 (250 mm × 2 mm) column by running only 12 mmol/L H₂SO₄without any organic solvent. This eluent not only effectively and isocratically separated all three catecholamines but also provided an acidic medium for CL reaction. This method appeared environmentally friendly and sensitive as no organic solvent was used to avoid quenching of CL signal, as a result, good LODs (0.6–5.1 mg/L) and recoveries (91.2% to 112.7%) were obtained with RSD less than 1.9% (Wu et al., 2012a, 2012b, 2012c, 2012d).

Naama Karu et al. investigated interactions between weak organic acid analytes (pharmaceutical-related compounds) including sodium benzoate, benzoic acid, trans-cinnamic acid, phenylacetic acid, benzenesulfonic acid, ibuprofen, mefenamic acid, naproxen, sulindac, 2-phenylbutyric acid, 1-naphthoic acid, 4-heptylbenzoic acid, 2-phenylsuccinic acid, and sodium 4-octylbenzenesulfonate. The assessment of analyte losses was carried out by comparison of UV absorbance before and after suppression at isosbestic wavelength. The obvious correlations were observed between analyte recovery rates after electrolytic suppression, eluent composition, suppression condition, and nature of analytes. It was also observed the hydrophobic adsorption interactions in the electrolytic suppressor is one of the main reasons to decrease analyte recovery, which is improved by adding the high content of acetonitrile. The organic solvent in the regeneration solution also prevents analyte permeation into the regeneration chamber through the suppressor membrane. In addition, the aging of electrolytic suppressor also caused analyte losses (Karu et al., 2012a, 2012b).

Shuchao Wu et al., fabricated an inline sensitive, fast, nonpolluting, and nontoxic post-column electrochemical derivatization setup and it was coupled with ion chromatography for isocratic separation and sensitive determination of non-fluorescent of folic acid (FA) and methotrexate (MTX) by using an anion exchange column. Both non-fluorescent analytes after online electrochemical oxidation turned into strong fluorescent products in optimum phosphate buffer solution (100 mM), which not only isocratically separated both FA and MTX but also worked as supporting electrolyte for their electro-oxidation. The developed method showed good linearity (0.01 mg L⁻¹ to 5 mg L⁻¹), the limit of detection (1.8 and 2.1 µg L⁻¹), and good recoveries in the range 80.2 to 103% in real plasma sample with a relative standard deviation less than 3.6% (Wu et al., 2012a, 2012b, 2012c, 2012d). The study was further extended for the separation determination of six phenols including 4-methyl phenol (pMP), 4-tert-butylphenol (TBP), 2, 4-dimethylphenol (DMP), 4- acetamidophenol (pAAP), 4-hydroxylphenolacetic acid (pHPA), and phenol (P) in real wastewater samples obtained from a refinery factory and a pool. All six phenols were isocratically separated on an IonPac AS11 column (4 mm i.d. × 250 mm) preceded by an IonPac AG11 guard column (4 mm i.d. × 50 mm) using NaOH eluent having as low acetonitrile content as 10% followed by post-column online electrochemical derivatization prior to fluorescence detection. It was observed that the electrochemical oxidation of respective phenol directly depends on potential applied for their conversion into fluorescent species, therefore potential was set 5.0 V till 4.5 min for phenol and pAAP and then brought back to 1.0 V for remaining analytes for their maximum conversion into fluorescent signals in basic media. The optimized method showed excellent LOD (0.4 µg/L and 3.8 µg/L), good linearity (2.0 – 1.0 × 104 µg/L for pAAP and DMP while 10 – 1.0 × 104 µg/L for P, pMP, pHPA, and TBP, respectively) with relative standard deviations were less than 4.8%. Finally, the developed method was successfully applied for the analysis of real samples, and the average recoveries of target spiked phenols were obtained in the range of 81.0–118% (Wu et al., 2012a, 2012b, 2012c, 2012d). The application of the designed system was further extended for the determination of carbamazepine (CBZ) in human plasma by using acidic media 0.1 M H₃PO₄ with a small content of ACN for its isocratic separation and online electrochemical derivatization into the fluorescent product. The developed IC-EC-FLD analytical method showed good LOD 0.3 mg/L with RSD less than 2.6%. It shows that this developed method is more sensitive as compared to IC coupled to ultraviolet detection (IC-UV). The method also showed excellent spiked recoveries (78.5 and 114%) for target drugs in two human samples (Wu et al., 2012a, 2012b, 2012c, 2012d).

Apart from anion exchange column, cation exchange column (SCX) has also been used for the analysis of alkaloids (Scopolamine, berberine, papaverine, boldine, brucine, noscapine, chelidonine, homatropine, ephedrine, hioscyamine, Pilocarpine) by varying the composition of eluent system by different type and concentration of buffers, different pH and different organic modifier methanol (MeOH), ACN, tetrahydrofuran (THF), dioxane (Dx). It was observed by increasing buffer concentration retention time of alkaloids decreased, theoretical plate number increased and peak symmetry improved whereas, only ACN and THF also helped to enhance the theoretical plate number and peak symmetry. Finally, the SCX column results were compared with those obtained by C18 column and appeared as the most efficient and selective system (Petruczynik and Waksmundzka-Hajnos, 2013).

Apart from these, chemometrics-assisted optimization, including response surface methodology and multivariate experimental design were utilized to develop anion-exchange liquid chromatography method for the sensitive determination of heparin and its impurities including over-sulfated chondroitin sulfate and dermatan sulfate. All analytes were separated on the IonPac® AS 22 column by using 2.5 M sodium chloride + 20 mM Tris phosphate buffer as an eluent for their gradient mode separation at a flow rate of 0.6 mL/min followed by UV detection at 215 nm. The analytical figures of merit were satisfactory with good linearity (r > 0.99), LOD (7.2–10.5 µg/mL) and relative standard deviations less than 11.4%. The method showed good recoveries in the range of 90.3 and 97.8% in real raw materials and heparin products (Thiangthum, et al., 2014).

Anna Petruczynik et al., proposed ion-exchange chromatography alternative to the reversed-phase (RP) chromatography for the separation of basic drugs. It was observed, the use of buffer mobile phase (potassium chloride and potassium phosphate) along with acetonitrile as an organic modifier entailed better efficiency and excellent symmetrical peaks for all drugs on the SCX stationary phase as compared to silica-based RP stationary phases, vulnerable to silanol effect. Therefore, this more efficient and selective IEC-UV method was further used for the determination of psychotropic drugs (escitalopram, sulpiride, zolpidem) in serum samples. The developed method showed good LOD 0.2166–0.2837 μg/mL and recoveries 71.9–98.6% (Petruczynik, et al., 2015).

Zhixiong Zhong et al., synthesized carboxylated graphene oxide/polyvinyl chloride (CGO/PVC) and its application as sorbent was investigated for the solid-phase extraction of selected sulphonamides antibiotics including sulfacetamide (SCT), sulfamonomethoxine (SMT), sulfamerazine (SMR), sulfadimethoxine (SDT), sulfadoxine (SDX), sulfanitran (SNT) and sulfaphenazolum (SPA) in cosmetics samples. The ion chromatographic separation was carried out by using IonPac® CS12A (250 mm × 4.0 mm i.d.) cation exchange column and 15 mM MSA + 28% (v/v) as eluent in isocratic mode, while all sulphonamides were detected at 270 nm by using a UV detector. The performance of synthesized sorbent was compared with commercially available ion-exchange, mixed-mode polymeric sorbent (WCX, MCX, C18). It was observed WCX and C18 gave poor recovery except for SMT, SCT, and SNT, while CGO/PVC sorbent had the better capacity and extraction selectivity to sulphonamides. The developed method showed good LOD (3.4–7.1 µg/L), recoveries (87.8–102.0%) with relative standard deviation values less than 6.4% (Zhong, et al., 2015).

There is a growing interest in the application of nanoparticles and nanotubes as nanosized SPE adsorbents for matrix elimination and enrichment of target analytes. A similar application of non-toxic and cost-effective SnO₂ NPs was investigated for the determination of non-fluorescent drug ketoprofen (KP) in human serum, urine, and canal water samples. The porous SnO₂ nanoparticles were synthesized by simple chemical precipitation method and their dual applications in ion chromatography were investigated; (i) for the conversion of non-fluorescent KP into fluorescent species and as an effective sorbent in micro-sample preparation for matrix elimination. The separation of KP from sample extract was carried out by using anion exchange column namely IonPacs® AG12A (50 mm × 4 mm i.d.; 13 mm p.s) coupled with IonPacs® AS12A (250 mm × 4 mm i.d.; 13 mm p.s) guard column, while 5 mM Na₂CO₃ having 10% (v/v) ACN was used as an eluent at the flow rate of 1.0 mL/min as shown in Fig. 6(a). Under same separation conditions, the interference study was also carried out by isocratic separation of KP in the presence of other four ubiquitously present pharmaceutical drugs namely ciprofloxacin, clofibrate, ibuprofen, and flurbiprofen as shown in Fig. 6(b). The confirmation of fluorescent ketoprofen was carried out via MS study. Fig. 6(c) shows that [M + H]⁺ appeared as the precursor ion at m/z 257.3 which corresponds to the fragment [M + H-C4H7O3]⁺ of KP. This approach appeared effective for the conversion of non-fluorescent analyte KP into respective fluorescent form for its sensitive and selective fluorescence determination. The developed analytical method showed satisfactory linearity (0.2–1.5 mg/kg), LOD (0.1–0.39 µg/kg), excellent recoveries (85.1–101.4%) with good intra-day and inter-day precisions (RSD < 16.3) for ketoprofen in three complex samples (Muhammad et al., 2017a, 2017b). This study was further extended for the determination of non-fluorescent antibiotics such as chloramphenicol from complex milk, human urine, and serum samples by utilizing porous SnO₂ nanoparticles as sorbent during a µ-QuEChERS sample preparation method. This sorbent not only helped to eliminate the matrix interferences but also converted this non-fluorescent antibiotic chloramphenicol into fluorescent species at the same time. On the optimization of various parameters such as NaBH₄ concentration, agitation temperature, optimization of sonication time, and selection of extracting solvent the developed method showed excellent linearity (r² ≥ 0.996) and LOD (0.0201–0.0280 μg/kg), satisfactory recoveries (78.3–100.2%) with inter- and intraday precisions ≤ 14.96%. On the successful application of this developed method for the determination of non-fluorescent antibiotic chloramphenicol from complex samples, it was further applied in order to generalize it for the conversion of non-fluorescent p-nitrophenol (p-NP) into fluorescent species for its sensitive determination with the help of cost-effective, simple and green IC-FLD technique (Muhammad et al., 2018a, 2018b, 2018c, 2018d).

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Fig. 6 (a) Separation of ketoprofen from human serum, urine, and canal water sample extract; (b) Chromatogram of isocratic separation of 1 mg/kg ciprofloxacin (CIP), clofibrate (CLO), ibuprofen (IBU), ketoprofen (KP), and flurbiprofen (FLU) by using 5 mM Na2CO3 + 10% ACN as eluent at the flow rate of 1.0 mL/min and; IonPacs AS12A column (250 mm × 4 mm i.d.; 13 mmparticle size) preceded by an IonPacs AG12A guard column (50 mm × 4 mm i.d.; 13 mm particle size); injection volume: 25 mL; and FLD detector wavelength: λ_ex/ λ_em for CIP = 295/340 nm, λ_ex/ λ_em for CLO & IBU = 229/329 nm, λ _ex/ λ _em for KP = 229/292 nm, and λ_ex/ λ_em for FLU = 235/315 nm; (c) Mass spectrum of the fluorescent ketoprofen with fragmentor potentials set at 100 V (Muhammad et al., 2018a, 2018b, 2018c, 2018d).

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4.1

4.1 Two-dimensional ion chromatography in pharmaceutical analysis

Analysis of target analytes in complex samples has been a challenging task due to the presence of interfering constituents, hence, in order to protect the analytical column from clogging along with suppression/enhancement of signals of target analyte, a suitable sample pre-treatment is indispensable. Since 1980, two-dimensional liquid chromatography (2D-LC) appeared a powerful technique for the analysis of such kinds of analytes of interest in complex matrices. This mode of analysis is quite useful in numerous ways such as carry-over, automation, better reproducibility, time-saving, to avoid sample loss and minimize the use of solvent and labor. However, limited applicability and stability to wide pH and lack of functionalities of silica based stationary phase in the fully aqueous mobile phase, force analyst to seek alternative and modified stationary phase to overcome above-stated problems (Iguiniz and Heinisch, 2017). Recently, ion chromatography appeared a better alternative for the analysis of polar and ionized organic analytes along with ions, carboxylic acid. Therefore, multiple review papers regarding the application of IC for the determination of organic analytes have been presented (Barron and Gilchrist, 2014, Fasciano and Danielson, 2016, Niemann and Anderson, 2008). The applications of advantageous IC are growing each decade for the determination of pharmaceutical drugs and related compounds. The IC evolved over time and appeared as an effective technique in the form of 2D-IC for the analysis of inorganic ions and polar organic analytes in various complex matrices. Besides, IC is also successfully being combined with other chromatographic techniques for this purpose (Brudin, et al., 2010). Zhu et al., coupled ion chromatography and ultrahigh performance liquid chromatography for simultaneous determination of inorganic anions and folic acid in folic acid tablets. A reverse phase C18 column was used as a pretreatment column on the first dimension for matrix elimination as well as for separation of inorganic ions from organic folic acid. These inorganic ions have no retention time on the reverse phase column, as a result these directly transfer onto the concentrating column and meanwhile folic was cleanly separated on this dimension and detected by UV detector at 280 nm. In the second dimension, all ions were isocratically separated on an IonPac® AS11-HC column (250 mm × 4 mm, 5 µm p.s) and detected by suppressed conductivity detector. This appeared as an effective technique for the simultaneous determination of inorganic and organic analytes with good linearity, low detection limit, and excellent recoveries with high reproducibility. In addition, this online sample preparation and analysis not only saves solvent, sample, and time but also can analyze inorganic anions and folates in complex matrices (Wang et al., 2018). The same analytical technique was further applied for the simultaneous separation and determination of inorganic ions (nitrite, nitrate, sulfite and sulfate, parabens) and four organic parabens including methylparaben (MP), propylparaben (PP), ethylparaben (EP) and butylparaben (BP) in cosmetics and for online elimination of complex matrix interferences. The proposed analytical method gave satisfactory results with good linearity (0.9994–0.9999), LOD (8.0 to 80 mg/L), excellent recoveries (93.9%-106.2%) in cosmetic samples (Wang et al., 2017a, 2017b). Similarly, it was successfully applied for the simultaneous determination of vanillin, ethyl vanillin, and six inorganic anions (i.e., BrO^{3 −}, NO²⁻, Cl⁻, NO^{3 −}, ClO³⁻, and SO₄²⁻) in complex food samples (Wang et al., 2017a, 2017b).

Due to the growing interest of two-dimensional chromatographic techniques; Ion exclusion chromatography and suppressed ion chromatography were combined for the determination of γ-hydroxybutyrate (GHB) in complex serum samples. High capacity Ion-exclusion column was fitted in the first dimension for matrix elimination and finally, matrix-free GHB separated on the ion-exchange column and excellent analytical figures of merit were obtained (Liu, et al., 2017). The same group introduced a powerful 2D-IC system for the simultaneous determination of five acidic pharmaceutical drugs viz. ibuprofen (IBU), clofibric acid (CLO), naproxen (NAP), flurobrofen (FLU) and aspirin (ASP) in complex sample extracts of apple, spinach, and hospital sewage sludge obtained by QuEChERS extraction. This system appeared simple, robust and flexible for the online matrix elimination in one dimension keeping acidic analytes of interest preserved by utilizing the strong electrostatic force of attraction between ionized acidic drugs and anion exchange column IonPac®AS11-HC stationary bed, followed by isocratic separation of all five drugs in second dimensions on IonPac® AS12 column which were detected by sensitive fluorescence detector as shown in Fig. 7(a). Interestingly, basic media not only helped for matrix elimination and their isocratic separation but also enhanced and turned non-fluorescent drug (ASP) into fluorescent species. The developed method showed good linearity (0.976–0.996), low LOD (0.024 µg/kg − 8.70 µg/kg) and excellent recoveries (81.17–112.5%) with RSDs less than 17.8% (Muhammad et al., 2017a, 2017b). This study was further extended for the analysis of four non-fluorescent acidic pharmaceuticals diclofenac (DCF), bezafibrate (BEZ), 2,4-dichlorobenzoic acid (2,4-DCB) and ketoprofen (KTP), and in human serum and oral fluid. A cost-effective homemade photochemically induced fluorimetric (PIF) setup was introduced prior to the fluorescence detector for the direct online conversion of non-fluorescent analytes into their respective fluorescent species. The first dimension of this 2D-IC system helped to eliminate unwanted matrix impurities of biological samples followed by the second dimension that isocratically separated these four non-fluorescent acidic drugs by using an anion exchange column IonPac®AS16A column (250 mm × 4 mm i.d; 13 μm p.s) as shown in Fig. 7(b). The developed method had shown good selectivity, sensitivity, excellent recoveries (88.68 – 102.14%), and LOD (0.35 – 8.10 μg/L) with no matrix effect (Muhammad, et al., 2019).

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Fig. 7 (a) Chromatogram of separation of standard solution of CLO (clofibric acid); IBU (ibuprofen), ASP (aspirin), NAP (naproxen), FLU (flurbiprofen) standard sample by IonPac® AS12A column (250 mm × 4 mm i.d; 13 mm p.s) preceded by an IonPac® AG12A guard column (50 mm × 4 mm i.d; 13 mm p.s) while running 8 mM Na₂CO₃ + 15% ACN as a mobile phase in isocratic mode, (b) structure and chromatograms of standard solution of 2, 4-DCB (2,4-dichlorobenzoic acid), BEZ (bezafibrate), KTP (ketoprofen) and DCF (diclofenac) by implying IonPacs® AG16 (50 mm × 4 mm i.d; 13 μm p.s) guard column coupled with IonPacs™ AS16 (250 mm × 4 mm i.d; 13 μm p.s) analytical column with 35 mM NOH having only 20% (v/v) ACN at flow rate of 1.0 mL/min (Muhammad et al., 2017a, 2017b, Muhammad, et al., 2019).

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Further application of IC in this area (analysis of pharmaceutical drugs) is summarized in Table 2.

Table 2 Application of IC-FLD/UV for the determination of pharmaceutical drugs and antibiotics in different matrices.

Drugs	Matrix analyzed	Sample preparation method	Analytical Technique Used	LOD	LOQ	Recovery	Ref.
Bisphosphonates (ethane-1- hydroxy-1,1-bis-phosphonic acid (EHDP), di-chloromethylene bisphosphonic acid (Cl MDP), di-fluoromethylene bisphosphonic acid (F MDP) and 4-amino-1-hydroxybutane-1,1-bisphosphonic acid (AMDP)	Biological samples	-----	IC-FLD via post column reaction with Al+3 – morin (2́,3, 4́, 5, 7-pentahydroxyflavone) solution; Mobile phase: 2 mM sodium benzoate mobile phase at pH 6.5 with flow rate of 2.0 mL/min; Column: Hamilton PRP-X100, I.D. 150 mm × 4.1 mm, 10 µm, 0.19 mequiv./g exchange capacity	4–6 ng/L	-----	-----	(Lovdahl and Pietrzyk, 1999)
Bisphosphonates (etidronate, clodronate, amidronate, and alendronate)	Tablets samples	Simple extraction	IC-DAD; Column: Phenomenex Phenosphere SAX (150 × 2.0-mm, 5 μm) column (Torrance, CA); Mobile phase: For etidronate and clodronate 20 mM sodium citrate having pH 3.6 with a flow rate of 0.3 mL/min and 50 μL injection volume, while for alendronate and pamidronate Mobile phase: sodium citrate buffer (20 mM, pH 4.6) at flow rate, a flow rate of 0.25 mL/min	50–100 µg/mL		97.12% −102.92%	(Fernandes et al., 2007a, 2007b)
Bisphosphonates (zoledronic, alendronic, pamidronic acids) and phosphoric acid, phosphorous acid	Pharmaceutical formulation	Simple sample prepration	IC-inverse UV: Column: IC-Pak Anion HR analytical column; Mobile phase: 3 mM Nitric acid with flow rate of 1.0 mL/ min	0.011–8.7 µg/mL	0.034–26.4 µg/mL	95.25–103.37%	(Zirojevic, et al., 2015)
L-ascorbic acid, nitrite, sulfite, oxalate, iodide and thiosulfate	Soft drink, river and ground water	Direct filtration and analysis	IC-FLD via post column reaction with cerium(IV); Mobile phase: 1.8 mM Na2CO3–1.7 mM NaHCO3 + 4 mM 1,3,5-benzene tricarboxylate was AS4A analytical column; 0.4 mM cerium(IV) in 0.15 M sulfuric acid at 0.3 mL/min flow rate used as post column derivatization reagent	7–72 µg/kg	23.3–239 µg/kg	100.7–1002.5%	(Miura, et al., 2002)
Seven Stibonic Acids	--------	--------	IC- photodiode array (PDA) and electrospray ionization mass spectrometric detection (ESI-MS). Column: 250X4.1 mm Hamilton PRP-X100 column; Mobile phase: 0.1 M NH4OAc solution at pH 9.0 with NH4OH;	0.3 µg/l	0.999 µg/L	------	(Simmons and McCloud, 2003)
Saccharin, acesulfame-K, sodium cyclamate	Soft drinks, candies, mints, gums, mouthwashes, pharmaceutical dosage	Erlenmeyer flask extraction	IC-UV and conductivity detector; Dionex AS4A anion Mobile phase : E1 (3 mM Na2CO3) at flow rate 2.0 mL/min,while E2 1.5 mM Na2CO3 at flow rate of 1.5 mL/min	-------	-------	94.9–109.4%	(Biemer, 1989)
sodium cyclamate, aspartame, Sodium saccharin, acesulfame-K, citric acid	Tabletop, sweeteners, fruit juice, carbonated drink	Simple extraction	IC-UV and conductivity detector; Column: Dionex IonPac AS4A-SC analytical column (250X4 mm I.D.); Eluent: 1.0 mmol/1 Na2CO 3 (El) and 12.5 mmol/1 Na2CO 3 (E2).	0.019–0.044 µg/ml	0.063–0.146 µg/mL	93–107%.	(Chen, et al., 1997)
Affeine, theobromine, theophyllin	foods and pharmaceutical	Simple extraction	IC-UV; Column and eluent: Dionex HPIC-CS3 cation exchange column with 100 mmol/ HCl Eluent, while Dionex OmniPac PAX-100 anion exchange column with column and 15 mmol/l KOH + 1% CAN as an eluent	0.03–0.2 µg/ml	0.099–0.66 µg/mL	87–103%.	(Chen, et al., 1998)
Dodecyl trimethyl ammonium chloride, tetra decyl trimethyl ammonium bromide, hexa decyl trimethyl ammonium chloride	Commercial lysing reagents		IC-UV, suppressed conductivity; Column: Dionex IonPack NS1, 10 µm, 250 X 4 mm; Mobile phase: (A) 10 mM MSA in ACN + water (50:50 v/v), (B) 10 mM MSA in ACN + water (80:20 v/v)				(Giovannelli and Abballe, 2005)
norfloxacin, ciprofloxacin, enoxacin	Pharmaceutical formulations, biological fluids.	Direct filtration and injection	IC-FLD; Column: Dionex OmniPac PAX-500 column; Eluent: 15 mmol/L H2SO4 and 35% methanol	50–105 µg/l	0.166–0.34 mg/L	100–104%	(Zhang, et al., 2007)
Thiocyanate	Plasma	SPE	IC-UV; Column: A Zorbax SAX C18 anion exchange column (250 × 4.6-mm i.d., 5-µm particle size); Mobile phase: 10 mmol/L phosphate buffer having pH 3.5 maintained by orthophosphoric acid.	2.5 mg/L	8.3 mg/L	93–103.9%	(Saussereau, et al., 2007)
Monoethylsulfate	Indinavir sulfate	Simple extraction	IC-UV/conduct metric; column: Metrosep A Supp5 (Metrohm, 250 mm × 4.0 mm 5 µm particle size); Eluent: 3.2mmole of sodium carbonate and 1mmole of sodium hydrogen carbonate	24 ng/ml	74 ng/mL	96.6–100.4%	(Prasanna, et al., 2009)
Zoledronic acid	Pharmaceutical Dosage	Simple extraction	IC-UV; Column: Allsep® anion exchange column, 150 X 4.6 mm i.d, 7 mm particle size; Mobile phase: 100% aqueous mobile phase contain only diluted formic acid at flow rate of 0.7 mL/min.	0.200–1.200 mg /mL	----------	----------	(Raghu, 2011)

5 Outlook

IC hyphenated to spectroscopic detectors (FLD or UV) appeared a great addition in chromatographic science. Further studies for the determination of analytes of other classes are still being carried out, which could further enhance the significance of hyphenated IC over HPLC for routine use. There is tremendous potential is available for further improvement in hyphenation of IC technique such as: (1) enormous interest and room have built up for the hyphenation of ion chromatography with other detectors such as atomic absorption spectroscopy (AAS), mass spectrometry (MS), photodiode array (PDA) and electrochemical detectors. Especially, MS has appeared as a powerful and widely accepted alternative for the selective and sensitive determination of analytes. Its effectiveness in the form of better sensitivity and selectivity can be further enhanced by coupling it with IC as its applications are growing each day in multiple fields (Barron and Gilchrist, 2014). Similarly, ICP/MS is generally a technique of the first choice for the selective and sensitive (up to ng/L) detection of elements. But ICP mostly uses aqueous phase and it is incompatible to chromatographic technique that uses high content of organic solvent such as acetonitrile in mobile phase. Therefore, IC coupled with ICP could be a perfect chromatographic alternative for the better separation and determination of analytes in both conditions of the mobile phase (Haddad, et al., 2008, Wang et al., 2017a, 2018, 2017b); (2) it would be interesting to investigate the physical and chemical impacts of pesticides and pharmaceutical drugs on aquatic life, fruits, food, and even plants apart from living species. Their trace concentration in various matrices could provide information for understanding the potential source and its remedies to control or minimize the contamination; (3) analytes at micro & nano level concentrations are more prone to living and non-living species (including humans) and can cause a greater and serious health risk. Therefore, more advanced, cost-effective, efficient, sensitive and miniaturized analytical techniques (i.e. IC-FLD) should be explored to analyze such kind of contamination in any sample matrix. There is a huge room available in the miniaturization of IC-FLD or other IC hyphenated techniques for its viable routine applications in food, forensics, omics, biological, environmental, and clinical fields. The miniaturized techniques are portable and use small volumes of sample (in micro or nanolitre) and organic solvents. It is expected to surmount various analytical challenges such as simplicity, reproducibility, accuracy, sensitivity, portability, environment friendliness (in terms of minimum consumption of organic solvent) robustness, onsite determination, and cost effectiveness for the routine analysis of organic and inorganic analytes of interest; (4) the new analytical and sample preparation should be developed within the “green” frame. Green Analytical Chemistry (GAC) approach will minimize the direct and indirect impact of analytical applications on the environment. There are various steps and ways through which this effect can be minimized such as eco-friendly eluent, online sample treatment, using online photochemical derivatization instead of complex toxic and laborious chemical derivatization, miniaturization of an instrument, miniaturization of sample preparation method with minimum processing steps, if possible direct analysis of the samples, using statistical modeling for assessing analyte contamination in targeted samples; (5) matrix effect (ME) is a serious limitation of chromatographic techniques especially when the spectrophotometric detector is coupled with these techniques for the determination of analytes in a complex sample matrix. ME can potentially suppress or enhance the signals of analytes of interest. This signal variation can cause a significant impact on the method LODs, repeatability, and even retention time of that particular analyte. Therefore, two dimensional (2-D) and three-dimensional hyphenated ion chromatographic approaches could solve the ME problem as compared to offline sample pre-treatment and one-dimensional IC–FLD analysis of multiple analytes in complex matrices (biological fluids, vegetable & fruit extracts and environmental samples). Besides, IC compatibility to the wide pH range (2–14) gives multiple choices to select the first dimension pre-treatment according to the nature of analytes and sample matrix (Hakme, et al., 2018, Losacco, et al., 2019, Rigano, et al., 2019); (6) the coupling of IC with nebulizing detectors such as mass spectrometer (MS), charged aerosol detectors (CAD) and evaporative light scattering detector (ELSD) is not common due to the use of buffer mobile phase in IC. The incompatibility of these detectors especially MS towards non-volatile content of the buffer mobile phase generally generates baseline noise and decreases the sensitivity of the instrument. Besides, the chances of salt precipitation are also high that could damage MS nebulizers and can clog the inlet. However, different non-suppressed hyphenated IC-system configurations can be adopted to minimize these effects such as; (i) diverting part of the mobile phase flow to the detector (ii) by adding the desalting device (iii) using volatile salts/organic solvents to the mobile phase. The IC mobile phase prepared with volatile salt such as ammonium hydroxide, ammonium nitrate, ammonium acetate, ammonium citrate, etc., can be a better choice for the application of IC-MS without using the desalting device. If this problem is resolved one way or another, this could be a huge success for the application of non-suppressed IC-MS for the analysis of organic analytes (pesticides and pharmaceutical drugs) in various samples such as beverages, polluted water, agricultural chemicals, food extract, and biological fluids (Karu et al., 2012a, 2012b, Karu, et al., 2011); and (7) miniaturization and automation of conventional sample preparation such as (solid‐phase extraction, pressurized liquid extraction coupled with solid‐phase extraction, liquid‐liquid microextraction, ionic liquid‐dispersive liquid‐liquid microextraction, hollow fiber liquid‐phase microextraction) with IC-FLD/UV could be a breakthrough in sample preparation. This mode of sample preparation can extensively reduce the matrix interferences, organic solvents, labor, time, sample loss, sample contamination, analyte degradation, and error as compared to offline extraction techniques. Furthermore, online sample treatment can improve the operation accuracy and make it a more efficient, sensitive, and selective alternative to offline methods (Li, et al., 2020, Muhammad, et al., 2019, Muhammad et al., 2017a, 2017b, Narenderan, et al., 2020, Nasiri, et al., 2020, Soriano, et al., 2019).

6 Advantages and limitation

Polymeric ion chromatography stationary phase has significantly solved the problem of pH tolerance, its rigidity, and resistance to organic solvents, unlike the reverse phase silica stationary phase. As a result of these characteristics, IC-FLD/IC-hv-FLD technique not only enhances the fluorescence intensity of most of the analytes in basic media but also helps to turn some of the non-fluorescent analytes into fluorescent form through online hydrolysis unlike the RP chromatography system, where an extra post-column pump is generally required for changing the column effluent pH for this purpose. In addition, ion exchange stationary phase separates different kinds of analytes such as the peptides, carbohydrates, nucleotides, pharmaceutical drugs, pesticides and polar, ionic or in the form of positional isomers (isomers, isobars) through electrostatic, hydrophilic and hydrophobic mode, which are difficult to separate and differentiate by the conventional reversed-phase stationary phase. In reverse phase chromatography, a small addition of organic solvent as a modifier in mobile phases normally changes or disturbs the retention order of polar or ionic analytes, whereas a small variation of modifier in IC eluent has a negligible or comparatively minimum effect on the retention order of same analytes. This is because the retention order of polar and ionic analytes is primarily defined by the buffer mobile phase concentration that controls the electrostatic force of attraction between the ion-exchange stationary phase and these polar or ionic analytes. This mechanism of separation in IC can help to significantly reduce the usage of organic solvent content, separation time along with a cost and simple way of analyte separation (Dogan, et al., 2020). Besides, polymeric IC columns are more durable and their monolithic forms can result in better separation of biomolecules and macromolecules (nucleic acids, peptides, proteins, and oligonucleotides) (Chankvetadze, et al., 2001, Fekete, et al., 2015, Karu et al., 2012a, 2012b, Lara, et al., 2013, Lucy, 2003, Muhammad, et al., 2019, Muhammad et al., 2017a, 2017b, 2018a, 2018b, 2018c, 2018d; Wang et al., 2018).

It is important to point out some major limitations observed till now because it is circumvented. For instance, it is challenging for any analytical technique to simultaneously separate highly hydrophobic analytes. Although, IC-FLD is a versatile, sensitive, selective and cost-effective technique that facilitates the detection of the compounds in µg/L range, but matrix effect has limited its most of the direct application for the analysis of complex samples. The ME can potentially entail inaccurate results due to potential enhancement or suppression of the target analyte signal. Therefore, an appropriate sample pretreatment is inevitable for the accurate determination of target analytes in complex sample matrices (Karu et al., 2012a, 2012b). One of the noticeable limitations of IC-FLD/UV is that it must rely on the retention times of analytes of interest for their identification and quantification. In complex samples, the risk of partial or complete matrix interference at the peaks of an analyte of interests are high, which can lead to misidentification of the peak along with inaccurate quantification.

Although IC-FLD appeared a simple, cost-effective, and sensitive analytical technique for the determination of pesticides and pharmaceutical drugs in various sample matrices. But there are many pesticides and pharmaceutical drugs that are not fluorescence active that is a huge barrier for their determination. Although IC-hv-FLD has overcome this problem to some extent by the online conversion of a number of aromatic non-fluorescent pesticides such as nicotinoids and drugs (nonsteroidal anti-inflammatory drugs) to their detectable forms. However, still there are groups of non-aromatic pesticides such as carbamate, pyrethroid, etc. that are not florescent active even upon online ultraviolet irradiation that has limited its direct widespread application (Muhammad, et al., 2019, 2018a, 2018b, 2018c, 2018d).

7 Comparison with other techniques

8 Conclusion

This review highlights the progress, development, advancement, and application of the hyphenated IC-FLD/UV technique for the analysis of pesticides and pharmaceutical drugs in different sample matrices. This technique is becoming increasingly vital for obtaining low LODs of large number of pesticides and pharmaceutical drugs that are present in traces in various complex matrices (biological fluids, vegetable & fruit extracts and environmental). Although, IC-FLD is a cost-effective, selective and sensitive technique but it is complementary to use appropriate sample preparation methods for the accurate, sensitive and selective determination of target analytes in complex sample matrices. Therefore, online sample pre-treatment techniques appeared a superior tool as compared to conventional off-line methodologies to eliminate maximum matrix interferences while minimizing analyte loss with better reproducibility and precision.

Matrix effect is not only observed in hyphenated IC but also in other analytical techniques such as LC-MS, GC–MS, which is needed to evaluate and upgrade to IC technique to nullify its effect for the accurate determination of pesticides and pharmaceuticals in complex matrices. We envision that online sample pretreatment techniques by using hyphenated IC technique will further evolve and provide an additional tool set to analyst for the determination of pesticides and pharmaceuticals in complex samples from biomedical, clinical, food and environmental fields.

Ethical approval

This article doesn’t contain any studies with human/animal participants. The authors confirm compliance with ethical standards.