INTRODUCTION

Separation techniques play a significant role in analysis, and chromatography is a robust separation method utilized in all research fields.[1] Chromatography passes a solution through a column filled with a suitable adsorbent, where the solutes are deposited in bands on the surface of a material. The bands move at different speeds when a pure solvent is introduced through the column.[2] Molecular properties linked to adsorption, partition, affinity, or discrepancies between their molecular weights are among the elements that impact this separation process. These variations lead specific mixture components to spend more time in the stationary phase and travel more slowly through the chromatographic system.[3] The word “chromatography” was first used in 1903 by the Russian botanist Mikhail Tswett. He employed liquid column chromatography, in which the mobile phase was a liquid and the stationary phase was a solid adsorbent loaded into a glass column. Using nearly 100 adsorbents, he studied chlorophyll extracts in petroleum spirit.[4-6] James and Martin documented the first analytical application of chromatography in 1952 when they used gas chromatography (GC) to analyze fatty acid compounds. There are several different forms of chromatography. Size, binding, affinities, charge, and other parameters are used in various chromatographic methods. Column chromatography, high-performance liquid chromatography (HPLC), GC, size exclusion chromatography, ion exchange chromatography are examples of different chromatography.[7]

HPLC is a type of liquid chromatography in which separation (or partition) happens between a mobile phase (the solvent) and a stationary phase (the column packing).[8] HPLC is widely utilized in qualitative and quantitative examination of many types of compounds. Validating a method is a crucial step in HPLC analysis. Determining whether an analytical technique is appropriate for the function is known as “analytical method validation.” Cost, simplicity, operator expertise, availability, and other factors are secondary to the actual validity of the approach under consideration when selecting an analytical method. During the validation phase, the following attributes are frequently tested: Specificity, robustness, linearity, precision, accuracy, limit of detection, limit of quantification, and solution stability.[9] HPLC is frequently used in the study of steroids since it offers an excellent tool for separation and quantification.[10] Reversed-phase mode separation is the preferred HPLC technique for all chemical classes. In reversed phase-HPLC, octadecyl silica (ODS or C18) columns are frequently employed as the stationary phase. Other materials can also be used to give various selectivity levels, including C8, C2, phenyl, amino, and cyano phases.[11] The chemistry of the mobile phase also affects selectivity. The mobile phase, which may be utilized in the isocratic or gradient mode, is typically prepared by mixing methanol or acetonitrile with varying amounts of water.[12] This paper summarizes the fundamental analytical criteria of HPLC such as column type, column temperature, mobile phase composition, flow rate, and detector type for 27 pesticides and 30 drug candidates.

APPLICATION OF HPLC IN PESTICIDES ANALYSIS

Pesticides, or antiparasitic chemicals used in agriculture, have quickly expanded in the past 30 years due to the advancement of organic synthetic chemistry.[13] Nowadays, over a hundred different pesticides are commonly used to protect plants. The problem of food contaminated with pesticides is a source of worry for practically everyone and everywhere. Several developed nations have implemented frequent monitoring programs for pesticide exposure control. These programs measure the extent of contamination in food items and highlight probable instances when pesticide residues surpass their tolerance thresholds due to poor farming practices.[14] Pesticide residues beyond the acceptable boundaries in vegetables during harvest are a significant cause for concern worldwide and nationwide.[15] The improper, wasteful, and unethical application of pesticides exacerbates the severity of the residue problem. Food products are dangerous for human consumption and export due to these residues. Furthermore, the residues harm the ecosystem.[16] As a result, applying more susceptible and selective analytical techniques to monitor pesticide residue quantities and regulate the biomagnification process is necessary due to the correspondingly enhanced intake of agrochemical pollutants into the environment. Much progress has been achieved in creating and utilizing various analytical techniques, including separation techniques such as GC and HPLC and detection methods such as electrochemistry, spectrophotometry, and spectrofluorimetry.[17] HPLC is increasingly used, particularly for the study of pesticides that GC cannot determine directly due to the compounds’ weak volatility, polarity, or thermal stability.[18] High-quality liquid chromatography with diode-array detection (DAD) can accurately identify pesticides in complex mixtures. These techniques have enabled detecting and quantifying pesticide residues in various atmospheres and food substances.[19-21] Creating a susceptible and highly accurate method is essential for accurately determining and measuring the analytes in complex matrices (such as food products). European Union directives specify the maximum residue levels for pesticides allowed in goods of plant or animal sources suitable for consumption by humans or animals.[22] With many pesticides in each analysis (injection), developing multi-residue technologies for pesticide analysis is crucial.[22] A comprehensive literature survey revealed that many solvents, including acetone or ethyl acetate, petroleum ether, n-hexane, and methylene chloride, have been employed to extract pesticide residue from fruits and vegetables. In this mini-review, we have summarization of 27 pesticide candidates [Table 1] with their chromatographic condition. The chromatographic information of the 27 pesticide candidates was collected from PubMed, NCBI, Google Scholar, Scopus, Web of Science databases.[23-42]

Table 1:: Summarization of pesticides with their chromatographic condition.

Pesticides Matrix Column Column temperature Mobile phase Flow rate Detector (nm) Ref. 2,4-Dichlorophenoxyacetic acid (2,4-D) Rat serum C18 40°C A=Acetonitrile
B=0.02 M ammonium acetate (containing 0.1% formic acid) 1.0 mL/min UV 230 [23] 3-Hydroxy carbofuran Coconut water C18 Room temperature A=Acetonitrile
B=Water 1.0 mL/min UV 275 [24] Carbofuran Coconut water C18 Room temperature A=Acetonitrile
B=Water 1.0 mL/min UV 275 [24] Acetamiprid Postmortem human blood, liver, stomach RP 80 40°C Acetonitrile: Water (50:50 v/v) 1.0 mL/min UV 248 [25] Alachlor Soils C18 60°C 25 mM dipotassium hydrogen phosphate
pH – 7.0: ACN
(80: 20 v/v) 1.0 mL/min UV 210 [26] Metolachlor Soils C18 60°C 25 mM dipotassium hydrogen phosphate pH – 7.0: ACN
(80: 20 v/v) 1.0 mL/min UV 210 [26] Aldicarb Vegetables and fruits C18 40°C A=Water
B=Acetonitrile 1.2 mL/min UV 210 [27] Aldicarb sulfone Vegetables and fruits C18 40°C A=Water
B=Acetonitrile 1.2 mL/min UV 210 [27] Aldicarb sulfoxide Vegetables and fruits C18 40°C A=Water
B=Acetonitrile 1.2 mL/min UV 210 [27] Benfuracarb Soil and water ODS Room temperature A mixture of acetonitrile-water (13: 7) 1.0 mL/min UV 280 [28] Benomyl Apple foliage ODS Room temperature ACN: H2O: Buffer (23:72:5% v/v) pH-7 0.8–1.5 mL/min UV 280 [29] Carbendazim Apple foliage ODS Room temperature ACN: H2O: Buffer (23:72:5% v/v) pH-7 0.8–1.5 mL/min UV 280 [29] Buprofezin Urine, serum, tomato, soil C18 25.0°C Acetonitrile: Buffer 75:25 (v/v) 1.0 mL/min UV 254 [30] Carbosulfan Oranges ODS 42°C Acetonitrile: Water 75:25 (v/v) 1.0 mL/min Fluorescence detector 330/465 [31] Diazinon Water and soil C18 Ambient temperature Acetonitrile: Water 65:35 (v/v) 1.0 mL/min UV 245 [32] Fenitrothion Water and soil C18 Ambient temperature Acetonitrile: Water 65:35 (v/v) 1.0 mL/min UV 245 [32] Dithianon Red pepper C18 35°C 1% AcOH in MeOH-H2O (60:40, v/v) 1.0 mL/min UV 263 [33] Fenarimol Blood, liver, and kidney samples C18 30°C Acetonitrile: Water 60:40 (v/v) 0.250 mL/min UV 225 [34] Hexaconazole Pesticide formulation C18 30°C A=ACN+MeOH (80+20)
B=Water (0.1% TFA) 60:40 (v/v) 1.0 mL/min PDA detector 205 [35] Imidacloprid Water and soil ODS 25°C Acetonitrile: Water 20:80 (v/v) 1.5 mL/min UV 270 [36] Lufenuron Napa cabbage C18 Room temperature Methanol: Water 75:25 (v/v) 1.0 mL/min UV 220 [37] Chlorfenapyr Napa cabbage C18 Room temperature Methanol: water 75:25 (v/v) 1.0 mL/min UV 220 [37] Metalaxyl-M Soil and sunflower plants Chiralcel OJ column Room temperature n-hexane:
2 propanol (15%v/v) 0.8 mL/min UV 254 [38] Oxadiazon Pesticide formulation C18 Room temperature Acetonitrile: Water 80:20 (v/v) 1.0 mL/min UV 292 [39] Pendimethalin Soil and garlic C8 Room temperature Acetonitrile: Water 80:20 (v/v) 1.0 mL/min UV 240 [40] Pyrazosulfuron-Ethyl Soils C18 30°C MeOH - H2O (0.2% Formic acid) 75:25 (v/v) 1.0 mL/min UV 241 [41] Sulfosulfuron Soils and wheat grain RP-8 Room temperature Acetonitrile: Water 80:20 (v/v)
Or
ACN: H2O: H3PO4
80:20:0.1 (v/v/v) 1.0 mL/min UV 212 [42] APPLICATION OF HPLC IN DRUG ANALYSIS

HPLC is a significant analytical technology used throughout the whole drug development, formulation, and manufacturing process in the newer pharmaceutical sector.[43] The use of liquid chromatography techniques in pharmaceutical analysis presents a potent weapon for clinical studies as well as pharmacological medication evaluation. Compared to earlier LC procedures, HPLC techniques have several benefits. They permit faster resolution time, better peak shapes, repeatable responses, and greater precision. HPLC columns do not need to be repackaged before use. Higher pressures can also be introduced to the solvent flow using HPLC columns.[44,45]

In the past 20 years, the rapid advancement of HPLC has allowed scientists to identify and quantify organic molecules, including pharmaceuticals and medication ingredients.[46] Scientists worked hard to discover a new method to fast-track their research. The drug industry tries to decrease research and innovation time and expenditures. For the development of chromatographic conditions, scientists tried to achieve their goal.[45] In this mini-review, we have summarization of 30 drug candidates [Table 2] with their chromatographic condition. The chromatographic information of the 30 drug candidates was collected from PubMed, NCBI, Google Scholar, Scopus, Web of Science databases.[47-75]

Table 2:: Summarization of drug with their chromatographic condition.

Drugs Column Column temperature Mobile phase Flow rate Detector (nm) Ref. Amoxicillin C18 Ambient Buffer: ACN (90:10% v/v) pH-7 1.0 mL/min UV 254 [47] Aprepitant C18 Ambient temperature Methanol: Water (90:10% v/v) 1.0 mL/min UV 220 [48] Cinitapride C18 Room temperature 0.1% HCOOH in H2O: ACN 0.5 mL/min UV 268 [49] Dexrabeprazole C18 Room temperature ACN: 0.025M KH2PO4 30:70 (v/v) 1.0 mL/min UV 284 [50] Dimenhydrinate C8 Room temperature 0.05M KH2PO4: Methanol (35:65, v/v) 1.0 mL/min DAD 240 [51] Diphenhydramine C18 Room temperature MeoH: ACN: H2O: 10mM Heptane sulfonate and 13 mM Triethylamine, (10:26:64) 1.0 mL/min UV 254 [52] Domperidone C18 Room temperature MeoH : KH2PO4 (65:35% v/v) pH-3 1.0 mL/min UV 227 [53] Esomeprazole C18 Room temperature ACN: Phosphate buffer (50:50% v/v) 1.0 mL/min UV 302 [54] Hydrocortisone C18 Room temperature MeoH: H2O: Acetic acid
(60: 30: 10, v/v/v) 1.0 mL/min UV 254 [55] Hyoscine C18 30°C A=0.01M K2HPO4 containing 2 g/L heptane sulfonic acid sodium salt, pH-3
B=Acetonitrile, 80% v/v 2.0 mL/min DAD 210 [56] Ilaprazole C18 Room temperature Methanol: Water (70:30% v/v) pH-3.0 1.0 mL/min UV 237 [57] Itopride C18 Room temperature A=Buffer 1.4 mL ortho-phosphoric acid at pH-3.0 with triethylamine
B=Acetonitrile 1.0 mL/min UV 220 [58] Lafutidine C18 Room temperature 0.02M K2HPO4: ACN (30:70, v/v) 1.0 mL/min UV 215 [59] Meclozine hydrochloride C8 Room temperature 0.2% triethylamine in water: Methanol (65:35, v/v) 1.0 mL/min PDA 229 [60] Mosapride C18 40°C Methanol: 0.02M K2HPO4 (70:30, v/v) 1.1 mL/min UV 274 [61] Omeprazole C18 40±1°C Phosphate buffer (pH 7.4): ACN
(70:30 v/v) 1.5 mL/min UV 280 [62] Prucalopride C18 Room temperature 0.1% H3PO4: MeoH (30:70 v/v) 1.0 mL/min UV 225 [63] Rabeprazole C18 Room temperature MeoH: H2O (65:35 v/v) 0.8 mL/min UV 284 [64] Donepezil C8 50°C Buffer: Methanol: Triethylamine
(55:45:5 v/v) 1.0 mL/min PDA 271 [65] Flavoxate C8 35°C ACN: MeOH: 0.1% HCOOH (5:20:75%v/v) 1.0 mL/min UV 311 [66] Homatropine C8 Room temperature ACN: Potassium dibasic phosphate 10 m Mol/L
PH--6.9
(35:65 v/v) 1.0 mL/min UV210 [67] Pilocarpine C18 25°C A=Phosphoric acid at pH-3.0 with triethylamine
B=MeOH (90:10 v/v) 1.0 mL/min DAD 215 [56] Carbimazole C18 Room temperature MeoH: 0.1% H3PO4
(80:20 v/v) 0.7 mL/min UV 291 [68] Hydrocortisone RP-column 40°C ACN: Buffer (75:25% v/v) 1.0 mL/min UV 254 [69] Pioglitazone C8 Room temperature ACN: 140mM KH2PO4 (40:60% v/v) 1.4 mL/min UV 269 [70] Azathioprine C18 Room temperature ACN: H2O (50:50% v/v)
pH-3.3 1.0 mL/min UV 276 [71] Cytarabine C18 Room temperature ACN: Buffer (Ammonium acetate) (30:70% v/v) 1.0 mL/min UV 272 [72] Melphalan C18 Ambient ACN: H2O: 1% H3PO4
(70:27:03%v/v) 1.0 mL/min UV 275 [73] Oxaliplatin C18 25±2°C 0.01 M phosphoric acid: Acetonitrile (95:05% v/v) 1.0 mL/min UV 255 [74] Vincristine C18 Ambient 0.02 M phosphate buffer, pH-5.4: Acetonitrile (50:50% v/v) 1.0 mL/min UV 233 [75] CONCLUSION

HPLC is a popular method for the analysis of pesticides and drugs. Determining pesticides is crucial because even minute amounts of a compound can be hazardous or detrimental to health. In drug analysis, HPLC is employed to find out pure compounds quickly. This review article helps researchers to know the chromatographic condition required for analyzing some common pesticides and drug molecules.