Use of Reversed-phase HPLC (RP-HPLC)

Introduction

Reversed-phase HPLC (RP-HPLC) is one of the commonly used techniques in separation of a wide ranging of analyte based on differences in their structures. In RP-HPLC, the polar mobile phase and nonpolar stationary phase was practised. In this mode of HPLC, the analyte retains into stationary phase by hydrophobic interaction (Hanai, 1999; Swadesh, 2001). The surface area of nonpolar part of analyte molecules is more readily to bind to the RP-HPLC stationary phase. Thus, the less polar analyte will has longer retention time, whereas polar analyte elute more readily.

1. Stationary phase and Column packing

In RP-HPLC, the stationary phase is employing of alkylated silica gel column. In other words, the column is packing of nonpolar & hydrophobic organic species (eg. octyl, octadecyl and phenyl groups). These hydrophobic organic species attached by siloxane bonds (-Si-O-Si-) to the silica surface (Corradini, Katz, Eksteen, Schoenmakers, & Miller, 1998). C18 and C8 phase are most commonly used in the column of RP-HPLC. C18 phase is more hydrophobic as it has longer alkyl chain length which bonded to silica gel. C8 has shorter alkyl chain length compared to C18. It has less carbon attached on silica gel. Besides, phenyl group that bonded to silica surface develops the – interaction of aromatic ring (Waksmundzka-Hajnos & Sherma, 2011).

2. Mobile Phase

The organic solvent such as methanol and acetonitrile are most widely used as mobile phase in RP-HPLC. It is due to they have the characteristic of free of particles and UV transparency (Swadesh, 2001). In addition, acetonitrile, methanol and tetrahydrofuran also served as organic modifiers in RP-HPLC. According to Hanai(1999), the concentration of organic modifiers could alter the overall retention time of analytes. However, the relative changes in retention time are still depends on the properties of analytes (Hanai, 1999; Waksmundzka-Hajnos & Sherma, 2011). Furthermore, isocratic and gradient elution also applied in RP-HPLC. In isocratic elution, mobile phase composition is remained constant throughout the elution. For gradient elution, the mobile phase composition is changed continuously throughout the elution. Gradient elution gives a better separation peaks for early eluters and sharper peaks for late eluters, but it need greater skills for method development (Ahuja & Dong, 2005).

3. Detector

There are many different spectrophotometric detectors that can be used in RP-HPLC. The main function of the detector in RP-HPLC is to trace and detect the presence of analyte components in the chromatography medium. These detectors are including ultraviolet-visible(UV-VIS) detector, diode array detector(DAD), chemiluminescence nitrogen detector(CLND), refractive index(RI) detector, mass spectroscopy(MS) and others (Swadesh, 2001). UV-VIS detector is the most popular detector among spectrophotometric detectors. Waksmundzka-Hajnos & Sherma (2011) stated that UV-VIS detector is utilized by referring to interaction of electromagnetic radiation with analyte sample at the wavelength in the region of 190nm to 1100nm. A high sensitivity of detector is crucial as it also control the signal noise level and drift of the baseline.

4. Applications

RP-HPLC methods mostly used to detect known or unknown substances in sample for the quality control. It is applicable in food chemistry, forensic chemistry, pharmaceutical chemistry, toxicological analyses, herbal drugs analyses and others.

2.0 Determination of drug samples by RP-HPLC

Reversed-phase High Performance Liquid Chromatography (RP-HPLC) has been generally and effectively utilised to determine numerous drugs samples.

Dual wavelength detectors have been employed in the simultaneous investigation of two antispasmodic drugs, phloroglucinol (PG) and its methylated derivative tri-O-methylphloroglucinol (TMP), that are established as pain-relieving drugs when used in combination as they manage to suppress the catechol-O-methyl transferase, relax the smooth muscles, and decrease the abdominal pain induced by glycerol. Present method employing RP-HPLC successfully separate and quantify PG and TMP by using isocratic elution and dual wavelength technique. Apart from studying the injectable sample solution, the serum extracted from blood of healthy volunteers and degraded compounds have also been investigated in order to obtain a complete analysis. Plasma was first centrifuged out of the blood, deproteinated and stock sample was added into the serum obtained. Degrading agent and stress conditions of hydrolysis were used in aid to degrade the PG and TMP. It has been investigated that only oxidation degraded the molecule drastically. (Hasan et al., 2013)

HPLC Conditions	Sensitivity (pg/mL)
Column = Hibar 𝜇Bondapak ODS C18 column Mobile phase = acetonitrile:0.005M sodium n-heptane sulphonate: 0.1M sulphuric acid (50 : 50 : 0.3, v/v/v) Flow rate = 1.0mL/min Detector wavelength = 266, 205 nm Injection volume = 20 𝜇L	LOD = 10 LOQ = 33

Cinitapride hydrogen tartarate has been also estimated by RP-HPLC. Cinitapride hydrogen tartarate is a new prokinetic drug, used as antiulcer agent of the benzamide by acting as an agonist of the 5-HT₁ and 5-HT₄ receptors and antagonist for 5-HT₂ receptors. The preparation of sample solution for this work is simple, by just powdered the tablets and mixed with the mobile phase used. The retention time obtained for cinitapride hydrogen tartarate is 3.737 min. Method validation and optimization have been carried out to validate the findings. (Reddy, Shekar & Murali, 2012)

HPLC Conditions	Sensitivity
Column = Inertsil ODS C18 column 150×4.6,5μ column Mobile phase = acetonitrile:phosphate buffer (30:70 v/v, pH 3) Flow rate = 1.0mL/min Detector wavelength = 264 nm Injection volume = 20 𝜇L	LOD = 1.410μg/ml LOQ = 4.700 pg/mL

Simultaneous investigation of lisinopril and non-steroidal anti-inflammatory drugs (NSAIDs) has been employed in bulk, pharmaceuticals formulations and human serum by RP-HPLC. The major uses of lisinopril are treating hypertension and congestive heart failure, preventing renal complications caused by diabetes while NSAIDs (naproxen, flurbiprofen, diclofenac sodium and mefenamic acid) are primarily used for treatment of acute or chronic pain and inflammation. As there might be interactions between lisinopril and NSAIDs, these two compounds must be detected simultaneously. (Sultana, Arayne, Siddiqui & Naveed, 2012)

HPLC Conditions	Sensitivity (ng/mL)
Column = Purospher star C18 (5μm,25×0.46 cm) column Mobile phase = methanol: water: acetonitrile (80:17.5:2.5 v/v, pH 3.0) Flow rate = 1.0mL/min Detector wavelength = 225 nm Injection volume = 20 𝜇L	Diclofenac sodium Flurbiprofen Lisinopril Mefenamic acid Naproxen	LOD 0.1 0.1 0.9 0.1 0.3	LOQ 0.4 0.5 2.8 0.3 1.1

Paracetamol, grouped in non-steroidal anti-inflammatory drugs has been discovered by colorimetric and spectroflurimetric techniques and being realised that it can combine with other drugs and thus, determined singly by RP-HPLC. It has antipyretic, analgesic and weak anti-inflammatory action, generally administered to suppress violent pains in advanced cancers. It has been found out that as concentration of one of mobile phases, ACN is high, paracetamol undergone improper dissolution. Meanwhile, phenomena such as broadening, fronting and tailing were remarkably lessened as ACN concentration gradually decreases. (Devi et al., 2013)

HPLC Conditions	Sensitivity (ng/mL)
Column = C18 column (4.6x250mm, pore size 5μm) Mobile phase = ACN : ultrapure water (25:75 v/v, pH 3.5) Flow rate = 1.0mL/min Detector wavelength = 207 nm Injection volume = 20 𝜇L	LOD = 120 LOQ = 360

Due to the rapid growth of demanding of NSAIDs, it is essential that to determine not the NSAIDs only, but also their combination drugs. The main function of NSAIDs is said to be inhibited cyclooxygenase in vitro and in vivo, hence decreasing the synthesis of prostaglandins which mediate the inflammation. Fifteen drugs have been simultaneously examined by robustness approach, including aceclofenac (ACF), aspirin (ASP), diclofenac (DCF), etoricoxib (ETC), ketorolac (KTL), paracetamol (PCM), salicylic acid (SA), ibuprofen (IBF) and naproxen (NPX) while the combination drugs being studied are clopidogrel (CLP), thiocolchicoside (THC), dextromethorphan (DXM), moxifloxacin (MXF), chlorpheniramine maleate (CPM) and domperidone (DOM). By varying the method parameters, effect on chromatographic separation of all the drugs can be investigated. (Patel, Samanthulam Shrigod, Modh & Chaudhari, 2013)

HPLC Conditions	Sensitivity (µg/mL)
Column = Kromasil C18 (250 × 4.6 mm, 5 𝜇m) Mobile phase = gradient of 15mM phosphate buffer (pH 3.25) and acetonitrile Flow rate = 1.1mL/min Detector wavelength = 230 nm Injection volume = 20 𝜇L	PCM THC SA MCX ASP DOM CPM DXM KTL ETC NPX ACF DCF IBF CLP	LOD 0.19 0.37 0.39 0.48 0.41 0.37 0.52 0.97 0.57 0.19 0.04 0.36 0.32 0.69 0.53	LOQ 0.64 1.22 1.3 1.61 1.36 1.24 1.75 3.24 1.9 0.65 0.14 1.18 1.05 2.3 1.77

Olmesartan medoxomil is an effective antihypertensive reagent, functioned as inhibitor that prevents the angiotensin II from binding to the AT₁ receptors in vascular muscle. The validated analytical method for the Olmesartan medoxomil determination in the presence of its degraded product in bulk drug has been established. The degraded products are formed under the conditions suggested by International Conference of Harmonization (ICH), which are acid hydrolysis (0.1M HCL), alkaline hydrolysis (0.1M NaOH), oxidation (30% H₂O₂), photolysis (UV), and thermal degradation under stress conditions. The sample was found to be highly susceptible to acid and alkaline hydrolysis and oxidation while for other conditions, no degradation was performed. (Hamrapurkar & Gadapayale, 2013)

HPLC Conditions	Sensitivity (µg/mL)
Column = C18 column (250×4.6 mm, 5 μm particle size) Mobile phase = methanol: water (60:40 v/v, pH 3.75) Flow rate = 1.0mL/min Detector wavelength = 270 nm Injection volume = 20 𝜇L	LOD = 0.02 LOQ = 0.06

Quantitative determination of oseltamivir phosphate (OSP) has been exercised by RP-HPLC. OSP is the drug to treat swine flu, prevents the virus from releasing by infected cells by selectively blocks the viral surface enzyme neuraminidase. Oseltamivir phosphate is also the drug of choice for treatment of avian influenza that diagnosed to be caused by H₁N₁ virus. This quantitative method was statistically validated for linearity, precision, accuracy, ruggedness, robustness and sensitivity. (Malipatil, Jahan & Patil, 2011)

HPLC Conditions	Sensitivity (µg/mL)
Column = Purosphere column (250mm x 4.6mm x 5.0μm) Mobile phase = ammonium acetate buffer and acetonitrile (60:40 v/v, pH 6.9) Flow rate = 1.0mL/min Detector wavelength = 220 nm Injection volume = 20 𝜇L	LOD = 0.495 LOQ = 1.5

3.0 Determination of food samples by RP-HPLC

Reversed-phase High Performance Liquid Chromatography (RP-HPLC) has been widely used to determine the food samples.

Honey is a food sources which consists of supersaturated solution of sugars (mostly fructose and sucrose) and other significant amount of minor compounds such as organic acids, furanic aldehydes and acids, enzymes, amino acids and proteins, mineral and water-soluble vitamins. The characterization of these minor compounds have been known to be a reliable tool to determine the botanical an geographical origin as well as the quality of the honey. Many methods have been launched out for the determination the minority organic compounds such as furanic aldehydes and acids but less for vitamins in honey. So, a new analytical method was proposed which utilize RP-HPLC to determine the presence of five water-soluble vitamins in honey that are vitamin B₂ (riboflavin), vitamin B₃ (nicotinic acid), vitamin B₅ (pantothenic acid), vitamin B₉ (folic acid) and vitamin C (ascorbic acid). Variety of validation parameters have been carried out in this proposed method in term of detection and quantification limit, linearity, precision, sensitivity as well as the bias to validate their findings (Ciululu et al., 2011).

HPLC Conditions	Sensitivity (mg/kg)
Column = Alltima C18 (5μm particle size, 250mm× 4.6 mm) column Mobile phase = Trifluoroacetic acid (0.025%, v/v) and acetonitrile Flow rate = 1.0mL/min Detector wavelength = 254nm for B3 and C vitamins; 210nm for B2, B5 and B9 vitamins Injection volume = 20 𝜇L	Vitamin B2 B3 B5 B9 C	LOD 0.25 0.25 0.58 0.15 0.10	LOQ 0.75 0.75 1.75 0.50 0.30

In fact, due to the higher concentrations of saccharides, slightly acidic condition and water activity as well as the presence of organic acids in honey, it favor the formation of furanic aldehydes especially 5-hydroxymethyl-2-furaldehyde (HMF). Hence, HMF is a good parameter for determine the quality of the honey. Apart from this, 2-furaldehyde, 2-furoic acid, 3-furaldehyde and 3-furoic acid also has been quantified in honey samples. Hence, 5-hydroxymethyl-2-furaldehyde (HMF), 2-furaldehyde (2-F), 3-furaldehyde (3-F), 2-furoic acid (2-FA) and 3-furoic acid (3-FA) has been investigated simultaneously and completely validated on 18 honey samples which different in their age, botanical and geographical origin by RP-HPLC. From the result, HMF was quantified in all samples. 2-F and 2-FA was showed in almost half of the samples whereas 3-F was detected in three honey samples and 3-FA in only one. Validation parameters were performed in term of detection limits, precision, linearity and accuracy (Spano et al., 2008).

HPLC Conditions	Sensitivity (mg/L)
Column = Alltima C18 (5μm particle size, 250mm× 4.6 mm) column Mobile phase = 0.1mol L-1 of H2SO4 in water and methanol Flow rate = 1.2mL/min Detector wavelength = 280nm until 11.5min, then 240nm Injection volume = 20 𝜇L	Analytes HMF 2-F 3-F 2-FA 3-FA	LOD 0.003 0.001 0.010 0.001 0.009	LOQ 0.010 0.004 0.030 0.004 0.027

Brominated phenols have been known to have strong odor properties and act as key flavor compounds in seafoods. The presence of the bromophenol compounds, their concentration and the marine environments controlled the different in the strength and characteristic of odor and flavor in seafoods. Hence, RP-HPLC was employed to determine the presence of simple bromophenols in marine fishes simultaneously which include 2-bromophenol (2-BP), 4-bromophenol (4-BP), 2,4-dibromophenol (2,4-DBP), 2,6-dibormophenol (2,6-DBP) and 2,4,6-tribromophenol (2,4,6-TBP). All the bromphenols have been extracted from the fish samples by combined steam distillation-solvent extraction (SDE) with 2mL of pentane/diethyl ether (6:4) and identified by RP-HPLC with UV-detection. (Silva et al., 2005)

HPLC Conditions	Sensitivity (ng/mL)
Column = LiChrospher 100 RP-18 (5μm particle size, 244mm× 4.4 mm) column Mobile phase = Gradient of water and acetonitrile Flow rate = 1.0mL/min Detector wavelength = 286nm for 2-BP, 4-BP, 2,4-DBP and 2,6-DBP; 297nm for 2,4,6-TBP Injection volume = 20 𝜇L	Analytes 2-BP 4-BP 2,4-BP 2,6-BP 2,4,6-TBP	LOD 127 179 89 269 232	LOQ 424 596 297 898 774

Nowadays, the employment of food additives in the production of processed and fast foods was gradually increasing throughout the world. However, illegal and excess addition of food additives can cause significant health problems were well known by community. For an example, in china, the ministry of Health of the People’s Republic of China strongly prohibited the addition of acesulfame, saccharin, neotame, stevioside, benzoic acid, caffeine and dehydroacetic acid as well as regulated the addition level of sorbic acid to 0.2g/kg into red wine. Variety of methods is available to determine the food additives present in food and drinks. However, simultaneous determination of large amount of food additives in red wine which mentioned as above was proposed by using dispersive solid phase extraction (dSPE) followed by RP-HPLC with ultraviolet (UV) detection in this study. The red wine samples were undergoes the dSPE methods which considered as most powerful cleanup technologies in removing various matrix and then analyzed by RP-HPLC with UV detection. Different kinds of validation parameters were utilized to determine the satisfaction and accuracy of the results. Apart from this, amino-functionalized Fe₃O₄ magnetic polymer (MP) which coupled with tetraethylenepentamine (TEPA) also proved that it is an efficient absorbent in dSPE extraction procedure which could eliminate most of the interferences in the red wine. (Zhao et al., 2013)

HPLC Conditions	Sensitivity (mg/L)
Column = Gemini C18 (5μm particle size, 250mm× 4.6 mm) column Mobile phase = 2.5mmol/L AmAc and 0.01% TFA (v/v) in water as solvent A; Acetonitrile as solvent B Flow rate = 1.0mL/min Detector wavelength = 210nm Injection volume = 10 𝜇L	Food additive Acesulfame Saccharin Caffeine Benzoic acid Aspartme Sorbic acid Stevioside Dehydroacetic acid Neotame	LOD 0.12 0.16 0.06 0.13 0.14 0.16 0.31 0.12 0.15	LOQ 0.35 0.49 0.17 0.40 0.41 0.49 0.92 0.37 0.46

Apart from this, the dosage of additives especially preservatives, is the most strictly controlled by EU law because of their potential risk to human health and safety. Nationally and international authorities have been established the guidelines for the usage of preservatives in food and foodstuff which about the maximum dosage, use conditions and type of food in which they can be used. In our daily life, the most common preservatives are sorbic acid, natamycin and lysozyme. So, analytical method for simultaneous determination of the four preservatives in different kinds of cheeses that are sorbic acid, natamycin, lysozyme and benzoic acid by single RP-HPLC was established. Benzoic acid was also included into the determination step is because it can be synthesized naturally from the microbial metabolism even it’s not added technically to cheese during production. All preservatives were extracted from the samples through a simple extraction step and then separated by RP-HPLC. Finally, the analytes were analyzed by a single wavelength UV detector (280nm) and even a triple wavelength UV detector (227nm, 280nm and 303nm) for a more sensitive determination. (Guarino et al., 2011)

HPLC Conditions	Sensitivity (mg/L) at =280nm
Column = Lichrosorb C18 chromatographic column (250mmx4.6mm, 5𝜇m particle size) coupled with C18 guard column (4x4mm, 5𝜇m) Mobile phase = 0.1% trifluoroacetic acid (TFA) in water (v/v) as eluent A, and 0.1% TFA in acetonitrile/tetrahydrofurane (5/1) (v/v) as eluent B. Flow rate = 1.0mL/min Detector wavelength = 280nm Injection volume = 20 𝜇L	Food additive Sorbic acid Benzoic acid Natamycin Lyzozyme	LOD 0.4 1.7 0.5 4.0	LOQ 1.3 5.6 1.8 13.3