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Application of HPLC in Biomedical Research for Pesticide and Drug Analysis
*Corresponding author: Bikash Debnath, Department of Pharmaceutics, Regional Institute of Pharmaceutical Science and Technology, Agartala, Tripura, India. bikashrips2014@gmail.com
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Received: ,
Accepted: ,
How to cite this article: Saha S, Mallik S, Debnath B, Singh WS, Ashif Ikbal A, Manna K. Application of HPLC in biomedical research for pesticide and drug analysis. Glob J Med Pharm Biomed Update 2023;18:20.
Abstract
Compared to traditional liquid chromatography, high-performance liquid chromatography (HPLC) delivers better results for analyzing unknown compounds. It permits faster resolution time, better peak shapes, repeatable responses, and greater precision. A comprehensive literature search has been conducted using online academic databases such as Google Scholar, PubMed, Web of Science, and Scopus, using keywords such as HPLC, pesticide analysis, drugs analysis, chromatographic conditions, and HPLC Column type. Total 75 number of articles were collected from peer-reviewed journals. With the help of literature review we have summarized the chromatographic condition of 30 drug candidates and 27 pesticide candidates. The study’s findings can guide future researchers to understand the chromatographic parameters of drugs and pesticides.
Keywords
High-performance liquid chromatography
Pesticides analysis
Drug analysis
Chromatographic conditions
Column type
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]
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]
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.
Declaration of patient consent
Patient’s consent not required as there are no patients in this study.
Conflicts of interest
There are no conflicts of interest.
Financial support and sponsorship
Nil.
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