Determination Of Different Classes Research Articles

Liquid chromatography with tandem mass spectrometric method for determination of 52 antibiotics in human whole blood and urine and application to forensic cases.

A rapid and reliable method was developed and validated for the simultaneous analysis of 52 antibiotics (cephalosporins, penicillins, carbapenems, lincosamides, quinolones, nitroimidazoles, macrolides, sulfonamides, tetracyclines, glycopeptide) in urine and whole blood by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Analytes were extracted by dilution or protein precipitation and analyzed on an Agilent 1260 HPLC system coupled to an Agilent 6470 Triple Quadrupole Mass Spectrometer. The method attended method validation criteria. The limits of detection were equal or lower than 2.0ng/mL, whereas the limits of quantification ranged from 0.1 to 10.0ng/mL, from 0.1 to 5.0ng/mL, in urine and whole blood, respectively. For all analytes, the bias and intra- and inter-day precision values were less than 14.7%. The ranges of recovery values of all antibiotics were 76.5-124.5% in whole blood and 76.3-121.8% in urine, values of the effects were lower than 25% in two matrices. No evidence of carryover was observed. The study of sample stability showed that almost all analytes were stable at 24°C for 24h, all analytes were stable at -20°C for 14 days and at -80°C for 30 days. Freeze-thaw cycles stability showed that antibiotics were stable except for imipenem. Autosampler stability study showed that all analytes were stable for 24h, except for imipenem and amoxicillin. Applicability was proven by analyzing authentic whole blood (n = 86) and urine (n = 79) samples from patients under antibiotics treatment. Therefore, this method was applied to the analysis 3 forensic allergy cases, which were positive for at least one analyte. A simple, sensitive and high-throughput method for the simultaneous determination of different classes of antibiotics in urine and whole blood samples was developed and applied. This sensitive method was successfully applied to forensic cases.

Derivatized versus non-derivatized LC-MS/MS techniques for the analysis of estrogens and estrogen-like endocrine disruptors in human plasma

Bisphenols, parabens, alkylphenols and triclosan are anthropogenic substances with a phenolic group that have been introduced to the environment in recent decades. As they possess hormone-like effects, they have been termed endocrine disruptors (EDs), and can interfere with steroid pathways in organisms. To evaluate the potential impact of EDs on steroid biosynthesis and metabolism, sensitive and robust methods enabling the concurrent measurement of EDs and steroids in plasma are needed. Of crucial importance is the analysis of unconjugated EDs, which possess biological activity. The aim of the study was to develop and validate LC-MS/MS methods with and without a derivatization step for the analysis of unconjugated steroids (estrone-E1, estradiol-E2, estriol-E3, aldosterone-ALDO) and different groups of EDs (bisphenols, parabens, nonylphenol-NP and triclosan-TCS), and compare these methods on a set of 24 human plasma samples using Passing-Bablok regression analysis. Both methods were validated according to FDA and EMA guidelines. The method with dansyl chloride derivatization allowed 17 compounds to be measured: estrogens (E1, E2, E3), bisphenols (bisphenol A-BPA, BPS, BPF, BPAF, BPAP, BPZ, BPP), parabens (methylparaben-MP, ethylparaben-EP, propylparaben-PP, butylparaben-BP, benzylparaben-BenzylP), TCS and NP, with lower limits of quantification (LLOQs) between 4 and 125 pg/mL. The method without derivatization enabled 15 compounds to be analyzed: estrogens (E1, E2, E3), ALDO, bisphenols (BPA, BPS, BPF, BPAF, BPAP, BPZ), parabens (MP, EP, PP, BP, BenzylP) with LLOQs between 2 and 63 pg/mL, and NP and BPP in semiquantitative mode. Adding 6 mM ammonium fluoride post column into mobile phases in the method without derivatization achieved similar or even better LLOQs than the method with the derivatization step. The uniqueness of the methods lies in the simultaneous determination of different classes of unconjugated (bioactive) fraction of EDs together with selected steroids (estrogens + ALDO in the method without derivatization), which provides a useful tool for evaluating the relationships between EDs and steroid metabolism.

Multiclass determination of endocrine disruptors in urine by hollow fiber microporous membrane and liquid chromatography

A simple and rapid methodology was developed using hollow fiber membrane microporous and a 96-well plate system for a high throughput multiclass determination of endocrine disruptors in human urine (diclofenac, diazepam, carbamazepine, ibuprofen, naproxen, carbofuran, methyl parathion, 17-α-ethynyl estradiol, bisphenol A and benzophenone). The quantification and detection of the chemicals were carried out by an HPLC-diode array detector. The fixed conditions for carrying out the method optimization were 1.5 mL of sample and 300 μL of solvent desorption. Multivariate and univariate models were applied to optimize the parameters of the method, achieving the following conditions: 20% diluted urine, 1-octanol of extraction solvent impregnated in the microporous membrane, 70 min extraction in pH 3.0 and 30 min with a mixture of 75% methanol and 25% acetonitrile (v/v) for the desorption. The R2 were ≤ 0.9973 for ibuprofen. The LOD ranged from 3.3 to 16.7 ng mL−1 and the LOQ from 10 to 50 ng mL−1. Relative recoveries ranged from 71% to 126%. The repeatability (n = 3) ranged from 0.22% to 12.01%, and the intermediate precision (n = 9) ranged from 0.13% to 17.76%. The method presents a good alternative for the determination of different classes of compounds in human urine.

A review on recent applications of deep eutectic solvents in microextraction techniques for the analysis of biological matrices

In general, the analysis of biological matrices is challenging and a sample preparation step employing extraction/cleanup techniques is required. Liquid-phase microextraction using deep eutectic solvents has been exploited in numerous applications because these solvents exhibit fascinating physico-chemical properties in addition to simple and straightforward preparation. Varied configurations and strategies have been examined for the determination of different classes of analytes, and these methodologies offer environmentally friendly workflows with low solvent and sample consumption, rapid and simple experimental procedures, and satisfactory analytical performance. In this review, developments related to the use of deep eutectic solvents in microextraction approaches for the analysis of biological matrices are briefly discussed with a focus on studies reported from 2017 to 2021.

Study of phenolic composition of olive fruits: validation of a simple and fast HPLC-UV method

A high-performance liquid chromatography (HPLC) method endowed with a gradient elution and a UV detection system was established and validated for the determination of phenolic acids, phenolic alcohols, hydroxycinnamic acid derivatives, flavonoids, secoiridoids and lignans during olive (Olea europaea L.) fruit development (green, purple and black olives). Within the test range, the calibration curves exposed a good linear regression (R 2>0.9995). Detection limits ranged between 0.63 and 13.43 mg/L for the detected phenolic compounds. The presented method yielded satisfactory repeatability in terms of retention times and average concentrations of phenolic compounds (RSD < 0.3%). Verbascoside was established as the major phenolic compound in black olives. Oleuropein was established as the dominating phenolic compound in green olives, and its level decreased during maturation. Additionally, this research is the first to experimentally evidence that the flavone luteolin-7-rutinoside is the predominant flavonoid glucoside in black olives, showing the most significant variation with fruit development. The above results validate the method for an easy and fast determination of different classes of phenolic compounds present in olive fruits.

Phytochemical Screening of Ficus benjamina (Linn.) Fruit Extracts

Ficus benjamina (Linn.) commonly called as weeping fig, is distributed throughout forests of eastern Himalayas of India, Fego, Travancore and Archipelago and China. It is useful in inflammation, syphilis, dysentery, erysipelas and as general tonic. Traditionally, fruit extract of Ficus benjamina has been used in skin disorders, inflammation, piles, leprosy, malaria, cancer, ulcer and as insect repellant. Detection, quantitative and qualitative determination of different classes of phyto-constituents present in any plant is considered important parameters which give an indication of the therapeutically active constituent(s) present in the plant. An extensive literature survey on Ficus benjamina showed that sporadic phytochemical reports are available. Therefore, it was considered worthwhile to perform phytochemical screening of various fruit extracts of Ficus benjamina which was not evaluated till date. Therefore, the present investigation was planned to investigate phytochemical screening of Ficus benjamina fruit extracts. Various extracts were prepared using successive Soxhlet extraction method. Solvents viz. petroleum ether, chloroform, ethyl acetate, ethanol and water in increasing polarity order were used to prepare extracts. Results of the present study showed that ethanol and ethyl acetate extract showed bioactive phytoconstituents like flavonoids, phenolic compounds and coumarins which could be responsible for various biological activities of Ficus benjamina.

Determination of features of cyber-attack goals based on analysis of data in open security data sources

The paper analyzes security databases including attack patterns database, vulnerability database and weaknesses database. Special attention is given to the schema that underlies the attack patterns database and specifies its objects and relations between them. This scheme is used for selection of features that characterize different classes of cyber-attack goals. The paper outlines metrics of security related objects, such as attacks, weaknesses and vulnerabilities provided by different schemas, the classes of cyber-attack goals, and analyzes relations between different goals and features of cyber-attacks. The experiments demonstrated dependency between the values of selected features and their applicability for determination of different classes of cyber-attack goals.

Rapid simultaneous determination of pentacyclic triterpenoids by mixed-mode liquid chromatography–tandem mass spectrometry

Pentacyclic triterpenoids (PCTs) possess high biological activity, including antitumor, anti-inflammatory, antiviral and hepatoprotective properties and are widespread in a plant biomass. Due to significant differences in polarity and other physicochemical properties, the simultaneous determination of different classes of PCTs by the methods of reversed phase liquid chromatography is difficult. In the present study, we proposed a new approach to chromatographic separation of such compounds based on the use of a stationary phase with a mixed retention mechanism combining hydrophobic, weak anion exchange and hydrophilic interactions. The use of the Acclaim Mixed-Mode WAX-1 column and tuning the selectivity by changing the contributions of different types of analyte–stationary phase interactions allowed the separation of 10 PCTs (betulin, erythrodiol, uvaol, friedelin, lupeol, β-amyrin, α-amyrin, betulinic, oleanolic and ursolic acids) belonging to four different classes (monools, diols, ketones and triterpenic acids) during 7.5 min in isocratic elution mode. The combination of this approach with atmospheric pressure chemical ionization tandem mass spectrometric detection and pressurized liquid extraction of analytes with methanol allowed to develop a rapid, accurate and highly sensitive method for analyzing PCTs in plant tissues with a total duration of the analytical cycle (including sample preparation steps) of not more than 40 min. It provides the detection limits in plant biomass extracts of 3–12 µg L–1 (44 µg L–1 for friedelin). The developed method was validated and successfully tested in the analyses of real birch bark and lingonberry peels.

Voltammetric Techniques for the Analysis of Drugs using Nanomaterials based Chemically Modified Electrodes

Background: Electroanalytical techniques play a very important role in the areas of medicinal, clinical as well as pharmaceutical research. Amongst these techniques, the voltammetric methods for the determination of drugs using nanomaterials based chemically modified electrodes (CMEs) have received enormous attention in recent years. This is due to the sensitivity and selectivity they provide on qualitative as well as quantitative aspects of the electroactive analyte under study. The aim of the present review was to discuss the work on nanomaterials based CMEs for the analysis of drugs covering the period from 2000 to present employing various voltammetric techniques for different classes of the drugs. Methods: The present review deals with the determination of different classes of drugs including analgesics, anthelmentic, anti-TB, cardiovascular, antipsychotics and anti-allergic, antibiotic and gastrointestinal drugs. Also, a special section is devoted for enantioanalysis of certain chiral drugs using voltammetry. The detailed information of the voltammetric determination for the drugs from each class employing various techniques such as differential pulse voltammetry, cyclic voltammetry, linear sweep voltammetry, square wave voltammetry, stripping voltammetry, etc. are presented in tabular form below the description of each class in the review. Results: Various nanomaterials including carbon nanotubes, graphene, carbon nanofibers, quantum dots, metal/metal oxide nanoparticles, polymer based nanocomposites have been used by researchers for the development of CMEs over a period of time. The large surface area to volume ratio, high conductivity, electrocatalytic activity and biocompatibility make them ideal modifiers where they produce synergistic effect which helps in trace level determination of pharmaceutical, biomedical and medicinal compounds. In addition, macrocyclic compounds as chiral selectors have been used for the determination of enantiomeric drugs where one of the isomers captured in the cavities of chiral selector shows stronger binding interaction for one of the enantiomorphs. Conclusion: arious kinds of functional nanocomposites have led to the manipulation of peak potential due to drug - nanoparticles interaction at the modified electrode surface. This has facilitated the simultaneous determination of drugs with almost similar peak potentials. Also, it leads to the enhancement in voltammetric response of the analytes. It is expected that such modified electrodes can be easily miniaturized and used as portable, wearable and user friendly devices. This will pave a way for in-vivo onsite real monitoring of single as well as multi component pharmaceutical compounds.

Geomorphometric analysis and terrain evaluation for environmental management in the Kurseong hill subdivision of the Darjeeling district, West Bengal, India

Kurseong hill subdivision of the Darjeeling district, West Bengal, is associated with complex geomorphic set-up stemmed from form–process interaction on varied slope, relief, elevation, aspects, ridge–valley orientation, density and frequency of drainage, dissection, ruggedness and topographic wetness over different stratigraphy of geological rocks having different hardness. This complexity provides ample scope to evaluate the terrain and categorize the terrain into different sensitivity zones according to land utilization point of view so that different terrain segments can be classified into different grades and therefore can be rationally utilized and managed for sustainable use. Therefore, the present paper takes an attempt to evaluate the terrain sensitivity of the Kurseong hill subdivision based on multi-criteria analysis method to identify different grades of terrain according to their ability to hold and sustain the pressure of land utilization for settlements, agriculture and other land uses. Terrain sensitivity evaluation of the Kurseong hill subdivision has been done on the basis of the study of hard rock geology, some selected geomorphometric parameters derived from 3-arc-second SRTM digital elevation data as well as SOI toposheets having RF of 1:50,000 and the existing land use factors after necessary ground truthing on some selected sites. The procedure adopted for multi-criteria analysis is 1 km2 grid-based rating value assignment algorithm of the selected parameters and determination of different classes of sensitive terrain and their gradation according to the capacity of the terrain to withstand present pressure of land utilization. Some measures for rational land utilization and management have also been proposed for optimal land use planning for the study area.

Development and Validation of a Hybrid Screening and Quantitative Method for the Analysis of Eight Classes of Therapeutants in Aquaculture Products by Liquid Chromatography–Tandem Mass Spectrometry

A method using reverse-phase ultra-high-performance liquid chromatography coupled with tandem mass spectrometry is described for eight classes of therapeutants that are used in marine aquaculture products. Validation studies to evaluate recovery, precision, method detection limits, and measurement uncertainty were performed at three levels, using three representative matrices [salmon (fatty fish), tilapia (lean fish), and shrimp (crustaceans)] to assess the method performance for use as a screening or determinative (quantitative and confirmatory) method. A total of 16 sulfonamides (plus 2 potentiators), 2 tetracyclines, 11 (fluoro)quinolones, 7 nitroimidazoles, 3 amphenicols, 5 steroids, and 3 stilbenes met the quantitative criteria for method validation. An additional 5 triphenylmethane dyes, 2 sulfonamides, 2 tetracyclines, and 1 amphenicol met the required performance for use as a screening method. Limits of detection (LODs) for the compounds that met the quantitative criteria ranged from 0.1 to 5 μg/kg, while LODs for compounds from the screening group ranged from 0.1 to 30 μg/kg. This method provides a comprehensive approach to the determination of different classes of compounds in aquaculture products.

Rapid and simple procedure for the determination of cathinones, amphetamine-like stimulants and other new psychoactive substances in blood and urine by GC–MS

In the last few years an increasing number of new psychoactive substances (NPS), with different chemical structures (of which 37% are stimulants), have been released into the illicit drug market. Their detection and identification in biological samples is hence of great concern.The aim of this work was to develop a high-throughput and rapid method for the determination of different classes of stimulants (amphetamine-type stimulants, cathinones, phenethylamines and ketamine analogues) from blood and urine samples using GC–MS.The proposed method allows the almost simultaneous derivatization and extraction of analytes from biological samples in a very short time, by using hexyl chloroformate as derivatization agent. The extraction of analytes was performed by Dispersive Liquid Liquid Microextraction (DLLME), a very rapid, cheap and efficient extraction technique that employs microliter amounts of organic solvents.The chromatographic method allowed for the separation of 26 stimulants including positional isomers (3-MMC and 4-MMC). The method was validated on urine and blood samples with the ability to detect and quantify all analytes with satisfactory limits of detection (LODs) ranging between 1 and 10ng/mL, limits of quantification (LOQs) between 2 and 50ng/mL, selectivity and linearity (5–1000ng/mL).The method was then applied to real samples from forensic cases, demonstrating its suitability for the screening of a wide number of stimulants in biological specimens.

Development and validation of a fully automated online-SPE–ESI–LC–MS/MS multi-residue method for the determination of different classes of pesticides in drinking, ground and surface water

ABSTRACTThe present work describes a fully automated method based on online solid phase extraction–liquid chromatography–tandem mass spectrometry for the determination of different classes of pesticides, including acidic and polar pesticides and six thiamethoxam metabolites. Sample preconcentration was performed by extracting 4 mL of the sample with a single styrene-divinylbenzene polymer. Elution of the compounds was done within the high performance liquid chromatography gradient and tandem mass spectrometry determination was performed in the selected reaction monitoring mode, by recording 1–3 transitions per compound. The overall pretreatment and analysis time per sample was less than 15 min. Method validation was performed in drinking, ground and surface water. For nearly all compounds a recovery between 70% and 120% could be achieved. The limit of detection ranges from 1.2 to 18 ng/L in drinking water and 3.0 to 23 ng/L in ground and surface water. The correlation coefficients for a calibration range of 0.05–2 µg/L are between 0.9915 and 0.9999. The limit of quantification (LOQ) for all compounds lies below the required limit of 0.1 µg/L, to fulfil the Council Directive 98/83/EC. Most of the compounds easily reach an LOQ below 0.05 µg/L.

Identification and determination of antibacterial substances in drugs by capillary electrophoresis

Methods of the quality control of finished dosage forms by capillary zone electrophoresis and micellar electrokinetic chromatography have been proposed. The optimum conditions for the separation and determination of different classes of antibacterial substances, penicillins, fluoroquinolones, nitrofurans, sulfanilamides, metronidazole, and chloramphenicol have been selected. The analytical range for the active ingredients of drugs was 1–1000 mg/g for solid and 0.001–0.50% for liquid drugs. The relative standard deviation of the results of analysis did not exceed 4%.

Optimization of an analytical methodology for the simultaneous determination of different classes of ultraviolet filters in cosmetics by pressurized liquid extraction–gas chromatography tandem mass spectrometry

A methodology based on pressurized liquid extraction (PLE) followed by gas chromatography–tandem mass spectrometry (GC–MS/MS) has been developed for the simultaneous analysis of different classes of UV filters including methoxycinnamates, benzophenones, salicylates, p-aminobenzoic acid derivatives, and others in cosmetic products. The extractions were carried out in 1mL extraction cells and the amount of sample extracted was only 100mg. The experimental conditions, including the acetylation of the PLE extracts to improve GC performance, were optimized by means of experimental design tools. The two main factors affecting the PLE procedure such as solvent type and extraction temperature were assessed. The use of a matrix matched approach consisting of the addition of 10μL of diluted commercial cosmetic oil avoided matrix effects. Good linearity (R2>0.9970), quantitative recoveries (>80% for most of compounds, excluding three banned benzophenones) and satisfactory precision (RSD<10% in most cases) were achieved under the optimal conditions. The validated methodology was successfully applied to the analysis of different types of cosmetic formulations including sunscreens, hair products, nail polish, and lipsticks, amongst others.

Simultaneous Determination of Different Classes of Pesticides in Breast Milk by Solid-Phase Dispersion and GC/ECD

This study presents the development, validation and application of a multiresidue analytical method intended to determine nine pesticides and a degradation product (α-endosulfan, β-endosulfan, α-HCH, g-HCH, aldrin, p,p'-DDE, p,p'-DDT, cypermethrin, deltamethrin, and trifluralin) in breast milk samples by solid-phase dispersion and gas chromatography with electron capture detector (GC/ECD). The proposed method is considered simple and efficient, combining extraction and clean up in a single step and quantification performed by standard addition to avoid the matrix effect. The method limits of detection and quantification varied between 0.002 and 0.079 µg mL-1 and 0.013 and 0.108 µg mL-1, respectively. The proposed method was applied to analyze 62 breast milk samples collected between February and June 2010 in Lucas do Rio Verde, Mato Grosso, Brazil. p,p'-DDE (0.32-12.03 µg g-1 of fat), p,p'-DDT (2.62-12.41 µg g-1 of fat) and β-endosulfan (0.54-0.61 µg g-1 of fat) were quantified in 29%, 5% and 3% of the samples, respectively. Aldrin was found below the method quantification limit in 7% of samples. Although these compounds were found in the analyzed samples, mothers were oriented to carry on feeding the infants as the breast milk is considered the optimum food during infancy.

Simultaneous quantification of several classes of antibiotics in water, sediments, and fish muscles by liquid chromatography-tandem mass spectrometry

Precise and sensitive methods for the simultaneous determination of different classes of antibiotics, including sulphonamides, fluoroquinolones, macrolides, tetracyclines, and trimethoprim in surface water, sediments, and fish muscles were developed. In water samples, drugs were extracted with solid-phase extraction (SPE) by passing 1000 mL of water through hydrophilic lipophilic balanced (HLB) SPE cartridges. Sediment samples were solvent-extracted, followed by tandem SPE (strong anion exchange (SAX) + HLB) clean-ups. Fish muscles were extracted by a mixture of acetonitrile and citric buffer (80:20, v/v) solution, and cleaned by SPE. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) with multiple reaction monitoring (MRM) detection was employed to quantify all compounds. The recoveries for the antibiotics in the spiked water, sediment, and fish samples were 60.2%–95.8%, 48.1%–105.3%, and 59.8%–103.4%, respectively. The methods were applied to samples taken from Dianchi Lake, China. It showed that concentrations of the detected antibiotics ranged from limits of quantification (LOQ) to 713.6 ng·L−1 (ofloxacin) in surface water and from less than LOQ to 344.8 μg·kg−1 (sulphamethoxazole) in sediments. The number of detected antibiotics and the overall antibiotic concentrations were higher in the urban area than the rural area, indicating the probable role of livestock and human activities as important sources of antibiotic contamination. In fish muscles, the concentration of norfloxacin was the highest (up to 38.5 μg·kg−1), but tetracyclines and macrolides were relatively low. Results showed that the methods were rapid and sensitive, and capable of determining several classes of antibiotics from each of the water, sediment, and fish matrices in a single run.

Determination of Aldoses, Deoxy-aldoses and Uronic Acids Content in a Pectin-Rich Extract by RP-HPLC-FLD after p-AMBA Derivatization

A reversed-phase high-performance liquid chromatographic method with fluorometric detection is proposed for the simultaneous determination of different classes of neutral sugars, such as hexoses (galactose, glucose and mannose), pentoses (arabinose and xylose), deoxy-hexoses (fucose and rhamnose), as well as acidic sugars (galacturonic and glucuronic acids). The separation is carried out on a hydrophilic end capped C18 column following a pre-column derivatization with p-aminobenzoic acid. The fluorometric detection of the derivatives has shown a strong dependency with the mobile phase pH. The performance of the proposed methodology was evaluated and the prerequisites of linearity (r-value > 0.999), precision (intra-day CV < 6 % and inter-day CV < 11 %) and recovery (between 77 ± 7 and 103 ± 3 %) were satisfied. To our knowledge, the obtained values of limit of detection for neutral sugars (within the range 6.1–28 μg L−1) are the lowest reported using this derivatizing agent. In order to better judge the methodology presented herein, neutral sugars of a pectin-rich orange extract were also analysed by the conventionally used GC-FID (gas-chromatography with flame ionization detector) method of alditol acetate derivatives. A statistical test (paired t test) has proved that no significant differences (α = 0.05) were observed between these two methods.

A method for the determination of multiclass pesticides in sugarcane juice employing QuEChERS and LC-ESI-MS/MS

This paper aims at describing a method for the determination of different classes of pesticides in sugarcane juice. The QuEChERS sample preparation method was used for the extraction of pesticides and their quantification was carried out by liquid chromatography-tandem mass spectrometry with an electrospray ionization source. After the optimization of the determination parameters of the pesticides, the method was validated by evaluating the analytical curves, linearity, limits of detection and quantification, precision (repeatability and intermediate precision), accuracy (recovery) and matrix effect. The detection limits of the method ranged from 0.83 to 16 μg L−1 and recoveries between 75% and 120% with an RSD lower than 20%. No pesticide residues were detected amongst the samples under analysis.

Quantitative Classification of DNA Damages Induced by Submicromolar Cadmium Using Oligonucleotide Chip Coupled with Lesion-Specific Endonuclease Digestion

Implementation of proper analytical tool for systematic investigation and quantitative determination of different classes of cadmium ion-induced DNA damages, especially at low metal ion concentrations, is still lacking. Using lesion-specific enzymes that cleave DNA at specific classes of damage and a fluorometric approach developed for quantifying fluorophore-labeled oligonucleotides bound to chip surfaces, we determined the frequencies of different lesions (strand breaks, oxidized purines, oxidized pyrimidines, or abasic sites) induced by submicromolar Cd(2+). Cd(2+)-treated oligonucleotide chips were digested with various endonucleases (Fpg protein, endonuclease III, endonuclease IV), producing a de novo single strand break (SSB) at their substrate modifications. The frequency of SSB and double strand break (DSB) was computed from the difference of pre- and post-Cd(2+)-treatment oligonucleotide coverage on the chip. While the frequency of SSBs and oxidized bases were successfully quantified even at 0.5 μM of Cd(2+), DSB frequency could be easily quantitated at 8.7 μM [Cd(2+)]. The numbers of abasic sites were below the oligonucleotide detection limit (2.4 amole; equivalent to 0.24 fM for a reaction volume of 100 μL). SSBs were found to constitute about 85-90% of single strand damages, while oxidized bases comprise only 4-7% of the total at 0.9 to 8.7 μM [Cd(2+)].