Amperometric cholesterol biosensor based on immobilized cholesterol esterase and cholesterol oxidase on conducting polypyrrole films

Fabrication of an amperometric cholesterol biosensor by co-immobilization of cholesterol esterase (ChEt) and cholesterol oxidase (ChOx) onto conducting polypyrrole (PPY) films using electrochemical entrapment technique is described. Electrochemical polymerization was carried out using a two-electrode cell configuration at 0.8 V. Characterization of resulting amperometric biosensor for the estimation of cholesterol has been experimentally determined in terms of linear response range, optimum pH, applied potential, temperature, and shelf-life. These PPY/ChEt/ChOx electrodes can be used for cholesterol ester estimation from 1 to 8 mM and have shelf-life of about 4 weeks at 4 °C during which about 15 estimations of cholesterol ester could be made. The sensitivity of PPY/ChEt/ChOx electrode has been found to be 0.15 μA/mM and the apparent K_m value for this electrode is 9.8 mM. Conductivity of the polymer films found to be about 3×10⁻³ S/cm.     

Zinc oxide nanoparticles-chitosan composite film for cholesterol biosensor

Zinc oxide nanoparticles (NanoZnO) uniformly dispersed in chitosan (CHIT) have been used to fabricate a hybrid nanocomposite film onto indium-tin-oxide (ITO) glass plate. Cholesterol oxidase (ChOx) has been immobilized onto this NanoZnO-CHIT composite film using physiosorption technique. Both NanoZnO-CHIT/ITO electrode and ChOx/NanoZnO-CHIT/ITO bioelectrode have been characterized using Fourier transform-infrared (FTIR), X-ray diffraction (XRD), cyclic voltammetry (CV), scanning electron microscopy (SEM) and electrochemical impedance spectroscopy (EIS) techniques, respectively. The ChOx/NanoZnO-CHIT/ITO bioelectrode exhibits linearity from 5 to 300 mg dl⁻¹ of cholesterol with detection limit as 5 mg dl⁻¹, sensitivity as 1.41 × 10⁻⁴ A mg dl⁻¹ and the value of Michaelis-Menten constant (K_m) as 8.63 mg dl⁻¹. This cholesterol biosensor can be used to estimate cholesterol in serum samples.     

Successive determination of urinary protein and glucose using spectrophotometric sequential injection method

A new sequential injection (SI) system with spectrophotometric detections has been developed for successive determination of protein and glucose. The protein assay is based on ion-association of protein with tetrabromophenolphthalein ethyl ester (TBPE) in the presence of Triton X-100 at pH 3.2. The blue product is monitored for absorbance at 607 nm. For glucose, hydrogen peroxide, generated by the oxidation of glucose in the presence of glucose oxidase immobilized on glass beads packed in a minicolumn, is monitored using iron-catalyzed oxidation reaction of p-anisidine to form a red colored product (520 nm). The SI procedure takes advantage in performing the protein assay during the incubation period for glucose oxidation. Linear ranges were up to 10 mg dL⁻¹ human serum albumin (HSA) with a limit of detection (LOD) (3σ) of 0.3 mg dL⁻¹, and up to 12.5 mg dL⁻¹ glucose with LOD of 0.08 mg dL⁻¹. R.S.D.s (n = 11) were 2.7% and 2.5% (for 1 mg dL⁻¹ and 5 mg dL⁻¹ HSA) and 1.4% (9 mg dL⁻¹ glucose). Sample throughput for the whole assay of both protein and glucose is 6 h⁻¹. The automated system has been demonstrated for the successive assay of protein and glucose in urine samples taken from diabetic disease patients, with good agreement with the other methods. This developed SI system is an alternative automation for screening for diabetic diagnosis.     

A novel paper-based device coupled with a silver nanoparticle-modified boron-doped diamond electrode for cholesterol detection

A novel paper-based analytical device (PAD) coupled with a silver nanoparticle-modified boron-doped diamond (AgNP/BDD) electrode was first developed as a cholesterol sensor. The AgNP/BDD electrode was used as working electrode after modification by AgNPs using an electrodeposition method. Wax printing was used to define the hydrophilic and hydrophobic areas on filter paper, and then counter and reference electrodes were fabricated on the hydrophilic area by screen-printing in house. For the amperometric detection, cholesterol and cholesterol oxidase (ChOx) were directly drop-cast onto the hydrophilic area, and H₂O₂ produced from the enzymatic reaction was monitored. The fabricated device demonstrated a good linearity (0.39 mg dL⁻¹ to 270.69 mg dL⁻¹), low detection limit (0.25 mg dL⁻¹), and high sensitivity (49.61 μA mM⁻¹ cm⁻²). The precision value for ten replicates was 3.76% RSD for 1 mM H₂O₂. In addition, this biosensor exhibited very high selectivity for cholesterol detection and excellent recoveries for bovine serum analysis (in the range of 99.6–100.8%). The results showed that this new sensing platform will be an alternative tool for cholesterol detection in routine diagnosis and offers the advantages of low sample/reagent consumption, low cost, portability, and short analysis time.     

Covalent immobilization of cholesterol esterase and cholesterol oxidase on polyaniline films for application to cholesterol biosensor

Cholesterol esterase (ChEt) and cholesterol oxidase (ChOx) have been covalently immobilized on electrochemically prepared polyaniline (PANI) films. These PANI/ChEt/ChOx enzyme films have been characterized using UV-visible, Fourier transform infrared (FTIR) spectroscopy and scanning electron microscopy (SEM). Electrochemical behavior of these films has been studied using cyclic voltammetry (CV) and amperometric techniques, respectively. The PANI/ChEt/ChOx enzyme films show broad oxidation peak from 0.2 to 0.5 V. These PANI/ChEt/ChOx biosensing electrodes have a response time of about 40s, linearity from 50 to 500 mg/dl of cholesterol oleate concentration. These PANI/ChEt/ChOx films are thermally stable up to 46 degrees C. This polyaniline based cholesterol biosensor has optimum pH in the range of 6.5-7.5, sensitivity as 7.5x10(-4) nA/mg dl and a lifetime of about 6 weeks.     

Tetraethylorthosilicate film modified with protein to fabricate cholesterol biosensor

Sol-gel derived tetraethylorthosilicate (TEOS) films were prepared by spin coating method on indium tin oxide (ITO) coated glass plate. Hydrophobic interaction method was used to coat the bovine serum albumin film over the surface of tetraethylorthosilicate sol-gel film to minimize cracking, biofouling and to improve the stability of the film. Cholesterol oxidase (ChOx) and horseradish peroxidase (HRP) were immobilized using covalent linkage with bovine albumin serum film to enhance the loading of the enzyme to improve the sensitivity of biosensor. Further ITO-TEOS-BSA-ChOx/HRP film was characterized by UV-vis, FTIR and SEM techniques. The optical response of the ITO-TEOS-BSA-ChOx/HRP biosensor was found to be linear in the range of 2-8 mM for cholesterol concentration with response time approximately 20 s. Amperometric response of ITO-TEOS-BSA-ChOx/HRP was observed to be linear in the range of 2-12 mM of cholesterol concentration with 10-s response time. Michaelis-Menten constant was calculated 21.2 mM .The shelf life of ITO-TEOS-BSA-ChOx/HRP biosensor was approximately about 8 weeks in desiccated conditions at room temperature.     

Langmuir-Blodgett film based on MEH-PPV for cholesterol biosensor

Cholesterol oxidase (ChOx) has been immobilized onto conducting poly[2-methoxy,5-(2′-ethyl-hexyloxy)-1,4-phenylene vinylene] (MEH-PPV)/stearic acid (SA) Langmuir-Blodgett film transferred onto octadecanethiol (ODT) modified gold plate. The ChOx/MEH-PPV/SA LB film bioelectrode exhibits has been characterized by FT-IR, contact angle, and atomic force microscopy. The response of the ChOx/MEH-PPV/SA LB film bioelectrode carried out using differential pulse voltammetry (DPV) studies reveal linearity from 1.29 to 12.91 mM of cholesterol concentration and response time as 30 s. This ChOx/MEH-PPV/SA bioelectrode exhibits values of correlation coefficient as 0.9939, standard deviation as 0.0029 μA and limit of detection as 1.66 mM. UV-visible spectrophotometer studies reveal that 5.2 × 10⁻³ U of ChOx are actively working per cm² area of ChOx/MEH-PPV/SA LB film bioelectrode and this bioelectrode is thermally stable upto 55 °C with reusability of about 60 times.     

Biosensor for total cholesterol estimation using N-(2-aminoethyl)-3-aminopropyltrimethoxysilane self-assembled monolayer

Cholesterol oxidase (ChOx), cholesterol esterase (ChEt), and horseradish peroxidase (HRP) have been co-immobilized covalently on a self-assembled monolayer (SAM) of N-(2-aminoethyl)-3-aminopropyltrimethoxysilane (AEAPTS) deposited on an indium–tin–oxide (ITO) glass surface. These enzyme-modified (ChOx-ChEt-HRP/AEAPTS/ITO) biosensing electrodes have been used to estimate cholesteryl oleate from 10 to 500 mg dL⁻¹. The sensitivity, K _m value, and shelf-life of these ChEt-ChOx-HRP/AEAPTS/ITO biosensing electrodes have been found to be 124 nA mg⁻¹ dL, 95.098 mg dL⁻¹ (1.46 mmol L⁻¹), and ten weeks, respectively. The ChEt-ChOx-HRP/AEAPTS/ITO bio-electrodes have been used to estimate total cholesterol in serum samples. Figure Covalent immobilization of enzymes onto AEAPTS/ITO surface using EDC/NHS chemistry Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Multienzymatic-rotating biosensor for total cholesterol determination in a FIA system

Fabrication of an amperometric-rotating biosensor for the enzymatic determination of cholesterol is reported. The assay utilizes a combination of three enzymes: cholesterol esterase (ChE), cholesterol oxidase (ChOx) and peroxidase (HRP); which were co-immobilizing on a rotatory disk. The method is developed by the use of a glassy carbon electrode as detector versus Ag/AgCl/3 M NaCl in conjunction with a soluble-redox mediator 4-tert-butylcatechol (TBC). ChE converts esterified cholesterol to free cholesterol, which is then oxidized by ChOx with hydrogen peroxide as product. TBC is converted to 4-tert-butylbenzoquinone (TBB) by hydrogen peroxide, catalyzed by HRP, and the glassy carbon electrode responds to the TBB concentration. The system has integrated a micro packed-column with immobilized ascorbate oxidase (AAOx) that works as prereactor to eliminate l-ascorbic acid (AA) interference. This method could be used to determine total cholesterol concentration in the range 1.2 μM-1 mM (r = 0.999). A fast response time of 2 min has been observed with this amperometric-rotating biosensor. Lifetime is up to 25 days of use. The calculated detection limits was 11.9 nM. Reproducibility assays were made using repetitive standards solutions (n = 5) and the percentage standard error was less than 4%.     

Amperometric choline biosensors prepared by layer-by-layer deposition of choline oxidase on the Prussian blue-modified platinum electrode

An amperometric choline biosensor was developed by immobilizing choline oxidase (ChOx) in a layer-by-layer (LBL) multilayer film on a platinum (Pt) electrode modified with Prussian blue (PB). 6-O-Ethoxytrimethylammoniochitosan chloride (EACC) was used to prepare the ChOx LBL films. The choline biosensor was used at 0.0 V versus Ag/AgCl to detect choline and exhibited good characteristics such as relative low detection limit (5 × 10⁻⁷ M), short response time (within 10 s), high sensitivity (88.6 μA mM⁻¹ cm⁻²) and a good selectivity. The results were explained based on the ultrathin nature of the LBL films and the low operating potential that could be due to the efficient catalytic reduction of H₂O₂ by PB. In addition, the effects of pH, temperature and applied potential on the amperometric response of choline biosensor were evaluated. The apparent Michaelis-Menten constant was found to be (0.083 ± 0.001) ×10⁻³ M. The biosensor showed excellent long-term storage stability, which originates from a strong adsorption of ChOx in the EACC multilayer film. When the present choline biosensor was applied to the analysis of phosphatidylcholine in serum samples, the measurement values agreed satisfactorily with those by a hospital method.     

Polypyrrole/multiwalled carbon nanotubes‐based biosensor for cholesterol estimation

The nanocomposite electrode comprising of polypyrrole (PPY) and carboxy functionalized multiwalled carbon nanotubes (MWCNT) has been electrochemically fabricated onto indium–tin–oxide (ITO) electrode using p‐toluene sulfonic acid (PTS). Cholesterol oxidase (ChOx) and cholesterol esterase (ChEt) have been immobilized onto this PPY– MWCNT/ITO nanocomposite electrode using N‐ethyl‐N‐(3‐dimethylaminopropyl) carbodiimide and N‐hydroxy succinimide chemistry for estimation of esterified cholesterol. The ChEt–ChOx/PPY–MWCNT/PTS/ITO bioelectrode has been characterized using Fourier transform infrared spectroscopy, electrochemical techniques, and scanning electron microscope. This ChEt–ChOx/PPY–MWCNT/PTS/ITO nanobioelectrode has a response time of about 9 s, linearity of 4 × 10^?4 to 6.5 × 10^?3 M/l of cholesterol oleate concentration, K_m of 0.02 mM, and thermal stability of upto 45°C. This electrode exhibits improved biosensing characteristics compared with other total cholesterol electrodes reported in literature till date and can be used to estimate cholesterol in blood serum samples. Copyright © 2011 John Wiley & Sons, Ltd.     

A blood-assisted optical biosensor for automatic glucose determination

A new approach for glucose determination in blood based on the spectroscopic properties of blood hemoglobin (Hb) is presented. The biosensor consists of a glucose oxidase (GOx) entrapped polyacrylamide (PAA) film placed in a flow cell. Blood is simply diluted with bidistilled water (150:1, v:v) and injected into the carrier solution. When reaching the PAA film, the blood glucose reacts with the GOx and the resulting H₂O₂ reacts with the blood Hb. This produces an absorbance change in this compound. The GOx-PAA film can be used at least 100 times. Lateral reactions of H₂O₂ with other blood constituents are easily blocked (by azide addition). The linear response range can be fitted between 20 and 1200 mg dL⁻¹ glucose (R.S.D. 4%, 77 mg dL⁻¹). In addition to the use of untreated blood, two important analytical aspects of the method are: (1) the analyte concentration can be obtained by an absolute calibration method; and (2) the signal is not dependent on the oxygen concentration.A mathematical model relating the Hb absorbance variation during the reaction with the glucose concentration has been developed to provide theoretical support and to predict its application to other compounds after changing the GOx by another enzyme. The method has been applied to direct glucose determination in 10 blood samples, and a correlation coefficient higher than 0.98 was obtained after comparing the results with those determined by an automatic analyzer. As well as sharing some of the advantages of disposable amperometric biosensors, the most significant feature of this approach is its reversibility.     

Accelerated solvent-based extraction and enrichment of selected plasticisers and 4-nonylphenol, and extraction of tin from organotin sources in sediments, sludges and leachate soils

Enrichment techniques have become an important feature in the trace analysis of oestrogen mimicking chemicals in the environment. Recent developments such as accelerated solvent extraction (ASE) have improved extraction recoveries in a wide variety of solid matrices including sediments, sludges and leachate soils. Such samples taken from the Irish Midlands Shannon Catchment region during the winter of 2004/5 and suspected to contain certain xenooestrogens or hormonally active agents were extracted using this technique, which was then coupled with high performance liquid chromatography (HPLC) for quantification purposes. ASE was thus employed to both isolate and pre-concentrate targeted analytes using the minimum amount of solvent hence making extractions more conservational. Two simple, yet extremely sensitive liquid chromatographic methods were developed based on UV detection; one for phthalates and one for alkylphenols, with recoveries reaching up to 92.0%. Acid digestion was used for the extraction of the tin and organotin compounds with analysis by polarography. In river sediment, levels of up to 24.4 mg kg⁻¹ phthalate, 1.14 mg kg⁻¹ 4-nonylphenol and 118 mg kg⁻¹ tin were found. In leachate sediments, values up to 49.8 mg kg⁻¹ phthalate, 1.57 mg kg⁻¹ 4-nonylphenol, and 36.0 mg kg⁻¹ tin were determined. In sludge, values up to 174 mg kg⁻¹ phthalate and 22.8 mg kg⁻¹ 4-nonylphenol were quantified. The highest value of tin (118 mg kg⁻¹) was found present in an area of high leisure craft activity. Typical sediment levels of tin at other river locations ranged between 1.20 and 37.5 mg kg⁻¹.     

Continuous glucose monitoring in interstitial fluid using glucose oxidase-based sensor compared to established blood glucose measurement in rats

Glucose monitoring is of importance for success of complex therapeutic interventions in diabetic patients. Its impact on treatment and glycemic control is demonstrated in large clinical trials. Up to eight blood glucose measurements per day are recommended. Notwithstanding, a substantial number of diabetic patients cannot or will not monitor their blood glucose appropriately. Considerable progress in control of disturbed metabolism in diabetic patients can be expected by continuous glucose monitoring. The aim of the study was to evaluate the performance of a new amperometric glucose oxidase-based glucose sensor in vitro and in vivo after subcutaneous implantation into rats.For in vitro testing current output of sensors was measured by exposure to increasing and decreasing glucose concentrations up to 472 mg dL⁻¹ over a time period of 7 days. After subcutaneous implantation of sensors into interscapular region of male rats glucose in interstitial fluid was evaluated and compared to glucose in arterial blood up to 7 days. Hyper- and hypoglycaemia were induced by intravenous application of glucose and insulin, respectively. Current of each implanted sensor was converted into glucose concentration using the first blood glucose measurement only.A change of current with glucose of 0.35 nA mg⁻¹ dL⁻¹ indicates high sensitivity of the sensor in vitro. The response time (90% of steady state) was calculated by approximately 60 s. Test strips for blood glucose measurement as reference for sensor readings was found as an appropriate and rapidly available method in rats by comparison with established hexokinase method in an automated lab analyzer with limits of agreement of +32.8 and −25.7 mg dL⁻¹ in Bland-Altman analysis. In normo- and hypoglycaemic range sensor readings in interstitial fluid correlated well with blood glucose measurements whereas hyperglycaemia was not reflected by the sensor completely when blood glucose was changing rapidly.The data given characterize a sensor with high sensitivity, long term stability and short response time. A single calibration of the sensor is required only in measurement periods up to 7 days. The findings demonstrate that the sensor is a highly promising candidate for assessment in humans.     

Dithiobissuccinimidyl propionate self assembled monolayer based cholesterol biosensor

A dithiobissuccinimidyl propionate (DTSP) self-assembled monolayer (SAM) prepared onto a gold (Au) surface has been utilized for covalent immobilization of cholesterol oxidase (ChOx) and cholesterol esterase (ChEt). These ChOx-ChEt/DTSP/Au bio-electrodes have been characterized using electrochemical impedance and cyclic voltammetric (CV) techniques, respectively. Differential pulse voltammetry (DPV) has been used for enzymatic assay of immobilized ChOx and ChEt onto the DTSP modified gold surface as a function of cholesterol oleate concentration. The response measurement conducted on ChOx-ChEt/DTSP/Au bio-electrode reveal the value of Michaelis-Menten constant (Km) as 0.95 mM suggesting enhanced affinity of enzymes (ChOx and ChEt). The ChOx-ChEt/DTSP/Au bio-electrodes show linearity in range of 50 to 400 mg dl(-1) of cholesterol oleate and the shelf-life of more than 50 days when stored at 4 degrees C. This biosensing electrode shows correlation coefficient of 0.9973 and standard deviation of regression as 0.859 microA.     

Application of octadecanethiol self-assembled monolayer to cholesterol biosensor based on surface plasmon resonance technique

Octadecanethiol (ODT) self-assembled monolayer (SAM) prepared onto gold-coated glass plate has been modified by using nitrene reaction of 1-fluoro-2-nitro-4-azidobenzene (FNAB) that further covalently binds to cholesterol oxidase (ChOx) via thermal reaction. FNAB acts as a bridge (cross-linker) between SAM and ChOx. The ChOx/FNAB/ODT/Au electrode thus fabricated has been characterized using contact angle (CA) measurements, UV-vis spectroscopy, electrochemical techniques and X-ray photoelectron spectroscopy (XPS) technique, respectively. This ChOx/FNAB/ODT/Au bioelectrode has been utilized for estimation of cholesterol in solution using surface plasmon resonance (SPR) technique. This SPR based cholesterol biosensor has linearity from 50 to 500 mg/dl of cholesterol in solution with lower detection limit of 50 mg/dl and shelf life of about 2 months when stored at 4 °C.     

Automatic flow system for the sequential determination of copper in serum and urine by flame atomic absorption spectrometry

In this work, a fully automated flow system exploiting the advantages of the association of multi-pumping, multicommutation, binary sampling and merging zones, to accomplish the sequential determination of copper in serum and urine by flame atomic absorption spectrometry, is described. The developed flow system allowed multiple tasks, such as serum samples preparation (samples and standard solutions viscosity adjustment), serum copper (SCu) measurement, urine copper (UCu) pre-concentration and its subsequent elution and measurement, to be carried out sequentially. The implemented flow manifold presented a modular configuration consisting on two quasi-independent modules, each one accountable for a specific sample manipulation and whose combined operation under computer control enabled the determination of copper in a wide concentrations range.Once optimised and with a sample consumption of about 0.250 mL of serum and 7 mL of urine, the developed flow system allowed linear calibration plots up to 5 mg L⁻¹ with a detection limit of 0.035 mg L⁻¹ for SCu and linear calibration plots up to 300 μg L⁻¹ with a detection limit of 0.67 μg L⁻¹ for UCu. The sampling rate varied according to the module employed and was about 360 determinations h⁻¹ (SCu module), 12 determinations h⁻¹ (UCu module) or 24 determinations h⁻¹ (12 urine and 12 serum samples; UCu and SCu modules simultaneously). Repeatability studies (R.S.D.%, n = 10) showed good precision for UCu at concentrations of 25 μg L⁻¹ (2.54%), 50 μg L⁻¹ (0.90%) and 100 μg L⁻¹ (1.62%) as well as for SCu at concentrations of 0.25 mg L⁻¹ (8.11%), 1 mg L⁻¹ (3.11%) and 5 mg L⁻¹ (0.90%). A comparative evaluation showed a good agreement between the results obtained in the analysis of UCu and SCu (n = 18) by both the developed methodology and the reference procedures. Accuracy was further evaluated by means of the analysis of reference samples (Seronorm™ Trace Elements Urine and Seronorm™ Trace Elements Serum) and the obtained results complied with the certified values.     

Electrochemical Cholesterol Sensor Based on Tin Oxide‐Chitosan Nanobiocomposite Film

A chitosan (CS)‐tin oxide (SnO₂) nanobiocomposite film has been deposited onto an indium‐tin‐oxide glass plate to immobilize cholesterol oxidase (ChOx) for cholesterol detection. The value of the Michaelis–Menten constant (K_m) obtained as 3.8 mM for ChOx/CS‐SnO₂/ITO is lower (8 mM) than that of a ChOx/CS/ITO bioelectrode revealing enhancement in affinity and/or activity of ChOx towards cholesterol and also revealing strong binding of ChOx onto CS‐SnO₂/ITO electrode. This ChOx/CS‐SnO₂/ITO cholesterol sensor retains 95% of enzyme activity after 4–6 weeks at 4 °C with response time of 5 s, sensitivity of 34.7 μA/mg dL^?1 cm² and detection limit of 5 mg/dL.     

Evaluation of alternate lines of Fe for sequential multi-element determination of Cu, Fe, Mn and Zn in soil extracts by high-resolution continuum source flame atomic absorption spectrometry

The usefulness of the secondary line at 252.744 nm and the approach of side pixel registration were evaluated for the development of a method for sequential multi-element determination of Cu, Fe, Mn and Zn in soil extracts by high-resolution continuum source flame atomic absorption spectrometry (HR-CS FAAS). The influence of side pixel registration on the sensitivity and linearity was investigated by measuring at wings (248.325, 248.323, 248.321, 248.329, and 248.332 nm) of the main line for Fe at 248.327 nm. For the secondary line at 252.744 nm or side pixel registration at 248.325 nm, main lines for Cu (324.754 nm), Mn (279.482 nm) and Zn (213.875 nm), sample flow-rate of 5.0 mL min⁻¹ and calibration by matrix matching, analytical curves in the 0.2-1.0 mg L⁻¹ Cu, 1.0-20.0 mg L⁻¹ Fe, 0.2-2.0 mg L⁻¹ Mn, 0.1-1.0 mg L⁻¹ Zn ranges were obtained with linear correlations better than 0.998. The proposed method was applied to seven soil samples and two soil reference materials (IAC 277; IAC 280). Results were in agreement at a 95% confidence level (paired t-test) with reference values. Recoveries of analytes added to soil extracts containing 0.15 and 0.30 mg L⁻¹ Cu, 7.0 and 14 mg L⁻¹ Fe, 0.60 and 1.20 mg L⁻¹ Mn, 0.07 and 0.15 mg L⁻¹ Zn, varied within the 94-99, 92-98, 93-101, and 93-103% intervals, respectively. The relative standard deviations (n = 12) were 2.7% (Cu), 1.4% (Fe - 252.744 nm), 5.7% (Fe - 248.325 nm), 3.2% (Mn) and 2.8% (Zn) for an extract containing 0.35 mg L⁻¹ Cu, 14 mg L⁻¹ Fe, 1.1 mg L⁻¹ Mn and 0.12 mg L⁻¹ Zn. Detection limits were 5.4 μg L⁻¹ Cu, 55 μg L⁻¹ Fe (252.744 nm), 147 μg L⁻¹ Fe (248.325 nm), 3.0 μg L⁻¹ Mn and 4.2 μg L⁻¹ Zn.