Development of a colloidal gold-based lateral-flow immunoassay for the rapid simultaneous detection of clenbuterol and ractopamine in swine urine

A multianalyte lateral-flow immunochromatographic technique using colloidal gold-labeled polyclonal antibodies was developed for the rapid simultaneous detection of clenbuterol and ractopamine. The assay procedure could be accomplished within 5 min, and the results of this qualitative one-step assay were evaluated visually according to whether test lines appeared or not. When applied to the swine urines, the detection limit and the half maximal inhibitory concentration (IC₅₀) of the test strip under an optical density scanner were calculated to be 0.1 ± 0.01 ng mL⁻¹ and 0.1 ± 0.01 ng mL⁻¹, 0.56 ± 0.08 ng mL⁻¹, and 0.71 ± 0.06 ng mL⁻¹, respectively, the cut-off levels with the naked eye of 1 ng mL⁻¹ and 1 ng mL⁻¹ for clenbuterol and ractopamine were observed. Parallel analysis of swine urine samples with clenbuterol and ractopamine showed comparable results obtained from the multianalyte lateral-flow test strip and GC-MS. Therefore, the described multianalyte lateral-flow test strip can be used as a reliable, rapid, and cost-effective on-site screening technique for the simultaneous determination of clenbuterol and ractopamine residues in swine urine.      

DNA Quantification in Nanoliter Volumes

A novel method for the determination of proteins at nanogram levels was proposed based on the decrease of resonance light scattering (RLS) signal resulting from the interaction of dibromo-o-nitrophenylfluorone (DBONPF)-sodium lauroyl glutamate (SLG) with proteins. At pH 2.97, the decrease RLS intensity was proportional to the concentration of proteins in the range of nanogram levels with 3σ detection limits being 3.4 ng mL⁻¹ for bovine serum albumin (BSA), 1.7 ng mL⁻¹ for human serum albumin (HSA), 4.1 ng mL⁻¹ for γ-globulin (γ-IgG), 4.4 ng mL⁻¹ for egg albumin, 6.2 ng mL⁻¹ for pepsin (Pep) and 3.7 ng mL⁻¹ for α-chymotrypsin (Chy). The method is no protein-to-protein variability, simple, rapid, practical and relatively free from interference from coexisting substance, as well as much more sensitive than most of the reported methods. The proposed method was successfully applied to determine total protein in human serum samples.     

Application of chitosan functionalized with 3,4-dihydroxy benzoic acid moiety for on-line preconcentration and determination of trace elements in water samples

Chitosan resin functionalized with 3,4-dihydroxy benzoic acid (CCTS-DHBA resin) was used as a packing material for flow injection (FI) on-line mini-column preconcentration in combination with inductively coupled plasma-atomic emission spectrometry (ICP-AES) for the determination of trace elements such as silver, bismuth, copper, gallium, indium, molybdenum, nickel, uranium, and vanadium in environmental waters. A 5-mL aliquot of sample (pH 5.5) was introduced to the minicolumn for the adsorption/preconcentration of the metal ions, and the collected analytes on the mini-column were eluted with 2 M HNO₃, and the eluates was subsequently transported via direct injection to the nebulizer of ICP-AES for quantification. The parameters affecting on the sensitivity, such as sample pH, sample flow rate, eluent concentration, and eluent flow rate, were carefully examined. Alkali and alkaline earth metal ions commonly existing in river water and seawater did not affect the analysis of metals. Under the optimum conditions, the method allowed the determination of metal ions with detection limits of 0.08 ng mL⁻¹ (Ag), 0.9 ng mL⁻¹ (Bi), 0.07 ng mL⁻¹ (Cu), 0.9 ng mL⁻¹ (Ga), 0.9 ng mL⁻¹ (In), 0.08 ng mL⁻¹ (Mo), 0.09 ng mL⁻¹ (Ni), 0.9 ng mL⁻¹ (U), and 0.08 ng mL⁻¹ (V). By using 5 mL of sample solution, the enrichment factor and collection efficiency were 8–12 fold and 96–102%, respectively, whereas the sample throughput was 7 samples/hour. The method was validated by determining metal ions in certified reference material of river water (SLRS-4) and nearshore seawater (CASS-4), and its applicability was further demonstrated to river water and seawater samples.     

Determination of vanadium(V) with CdTe quantum dots as fluorescent probes

CdTe quantum dots (QDs) were modified with thioglycolic acid (TGA) and synthesized in aqueous medium. The optimum fluorescence intensity was found to be at pH 6.24 with a CdTe QDs concentration of 4.96 × 10⁻⁷ mol L⁻¹. The quenched fluorescence intensity of CdTe QDs is linearly proportional to V(V) concentration from 10 to 200 ng mL⁻¹ with correlation coefficient R = 0.9985. The limit of detection for V(V) was 2.07 ng mL⁻¹. The proposed method was successfully applied to the analysis of trace amounts of V(V) in water samples with recovery of 96.5–101.8%, and the results were in good agreement with those of electrothermal atomic absorption spectrometry.     

Determination of trace rhodium by supramolecular catalytic kinetic spectrofluorimetry of β-CD-rhodium-KBrO3-vanillin salicylhydrazone

A sensitive catalytic kinetic spectrofluorimetric approach for determining ng mL⁻¹ levels of rhodium is presented, and the possible mechanism of the catalytic reaction was investigated. The determination is based on the catalytic property of rhodium to enhance the reaction of o-vanillin salicylhydrazone (OVSH) with potassium bromate in a water-ethanol medium at pH 4.80 and 45 °C. The presence of β-cyclodextrin (β-CD) obviously sensitized the assay due to its high inclusion ability towards OVSH. Under optimized experimental conditions, fluorescence measurements of the β-CD-rhodium-KBrO₃-OVSH catalytic kinetic reaction system were carried out in its fluorescent band centered at λ_ex = 333 nm and λ_em = 476 nm, respectively. The calibration graph was linear over the concentration range of 0.47–100 ng mL⁻¹ with a detection limit of 0.14 ng mL⁻¹. The effect of interferences was discussed, and the results show that the extraction method can be used to separate rhodium from interference species such as iridium. The proposed method, applied to several synthetic mixtures containing rhodium mixed with varying amounts of metal salts, produced satisfactory results.     

Simple and sensitive assay for nucleic acids by use of the resonance light-scattering technique with the anionic dye methyl blue in the presence of cetyltrimethylammonium bromide

This is the first report on the determination of nucleic acids based on the enhancement of resonance light scattering (RLS) of the anionic dye methyl blue (MB) in the presence of cetyltrimethylammonium bromide (CTMAB). In tris(hydroxymethyl) aminomethane buffer of pH 9.0, MB and nucleic acids react with CTMAB to form large particles of complex, which results in strong enhanced RLS signals characterized by three peaks at 334 nm, 393.5 nm and 548 nm. Mechanistic studies show that the enhanced RLS stems from the aggregation of MB on nucleic acids through the bridged and synergistic effect of CTMAB. With the enhanced RLS signals at the best wavelength at 334 nm, the enhanced RLS intensity is proportional to the concentration of nucleic acids in a wide range. The lowest limit of determination was 2.1 ng mL⁻¹, three synthetic samples were analyzed satisfactorily.     

Chemiluminescence of Peracetic Acid in Alkaline Medium and its Application to Dihydralazine Sulfate Determination

The chemiluminescence (CL) of peracetic acid (PAA) in alkaline medium is very weak but is strongly enhanced after the addition of dihydralazine sulfate (DHZS). Based on this phenomenon, a simple, rapid and highly sensitive flow-injection CL method for the determination of DHZS was developed. The CL emission was linearly related to the DHZS concentration in the range of 20–4000 ng mL⁻¹ with a detection limit (3σ) of 1.2 ng mL⁻¹. As a preliminary application, the proposed method was successfully applied to the determination of DHZS in pharmaceutical preparations; the recovery of DHZS in human urine was between 96.5% and 102.2%. A detailed CL mechanism was proposed and singlet molecular oxygen (¹O₂) was suggested to be produced in the CL reaction process.     

Determination of salbutamol using R-phycoerythrin immobilized on eggshell membrane surface as a fluorescence probe

A fluorescence sensor was fabricated using R-phycoerythrin (R-PE) immobilized on eggshell membrane as the fluorescence probe, and salbutamol was determined based on the decrease in fluorescence intensity of R-phycoerythrin. The scanning electron and fluorescence micrographs showed the microstructure of the eggshell membrane and indicated that the R-PE was successfully immobilized on the eggshell membrane surface. The effects of some experimental parameters on the response of the biosensor were investigated in detail. The fluorescence sensor has a linear response to salbutamol concentrations ranging from 5.00 to 100 ng mL⁻¹. The detection limit for the salbutamol is 3.50 ng mL⁻¹ (S/N = 3). The reproducibility of fabricating the biosensors using six different membranes was good with a relative standard deviation (RSD) of 3.28%. The fluorescence sensor showed extremely good stability with a shelf life of at least 50 days and reversible response to salbutamol. Some common potential interferents showed little effect on the response of the salbutamol fluorescence sensor. The proposed method was successfully applied to the determination of the salbutamol in urine samples.     

Comparison of Pyrolysis and Microwave Acid Digestion Techniques for the Determination of Mercury in Biological and Environmental Materials

 Microwave digestion reduction-aeration and pyrolysis combined with cold vapour atomic absorption and cold vapour atomic fluorescence are compared for the determination of total mercury in several biological and environmental matrices. The biological samples were digested in a mixture of HNO₃/H₂O₂, the environmental samples in a mixture of HNO₃/HClO₄. After reduction with SnCl₂, the mercury was collected by two-stage gold amalgamation. After microwave digestion reduction-aeration, detection limits of 1.4 ng g⁻¹ and 0.6 ng g⁻¹ were obtained for cold vapour atomic absorption spectrometry (CVAAS) and cold vapour atomic fluorescence spectrometry (CVAFS), respectively, for 250 mg of environmental samples. For biological samples (500 mg) the detection limits were 0.7 ng g⁻¹ (CVAAS) and 0.4 ng g⁻¹ (CVAFS). After pyrolysis, detection limits of 3.5 ng g⁻¹ and 1.6 ng g⁻¹ for CVAAS and CVAFS, respectively, were obtained for a 10 mg sample. Pyrolysis can only be applied when the organic content of the sample is not too high. Accurate results were obtained for 8 certified reference materials of both environmental and biological origin. In addition, a real sludge sample was analysed. Author for correspondence. E-mail: richard.dams@rug.ac.be Received September 18, 2002; accepted December 3, 2002 Published online May 5, 2003     

Extraction and determination of chloramphenicol in feed water,milk, and honey samples using an ionic liquid/sodium citrate aqueous two-phase system coupled with high-performance liquid chromatography

A green, simple, non-toxic, and sensitive sample pretreatment procedure coupled with high-performance liquid chromatography (HPLC) was developed for the analysis of chloramphenicol (CAP) that exploits an aqueous two-phase system based on imidazolium ionic liquid (1-butyl-3-methylimidazolium tetrafluoroborate, [Bmim]BF₄) and organic salt (Na₃C₆H₅O₇) using a liquid–liquid extraction technique. The influence factors on partition behaviors of CAP were studied, including the type and amount of salts, the pH value, the volume of [Bmim]BF₄, and the extraction temperature. Extraction efficiency of the CAP was found to increase with increasing temperature and the volume of [Bmim]BF₄. Thermodynamic studies indicated that hydrophobic interactions were the main driving force, although electrostatic interactions and salting-out effects were also important for the transfer of the CAP. Under the optimal conditions, 90.1% of the CAP could be extracted into the ionic liquid-rich phase in a single-step extraction. This method was practical when applied to the analysis of CAP in feed water, milk, and honey samples with a linear range of 2~1,000 ng mL⁻¹. The method yielded a limit of detection of 0.3 ng mL⁻¹ and a limit of quantification of 1.0 ng mL⁻¹. The recovery of CAP was 90.4–102.7% from aqueous samples of real feed water, milk, and honey samples by the proposed method. This novel process is much simpler and more environmentally friendly and is suggested to have important applications for the separation of antibiotics.     

Development of a cloud point extraction and preconcentration method for silver prior to flame atomic absorption spectrometry

The possibility was investigated of using 2-mercaptobenzothiazole (MBT) for Ag(I) concentration by micellar extraction at cloud point (CP) temperature and subsequent determination by flame atomic absorption spectrometry (FAAS). The method is based on the complexation of Ag(I) with 2-mercaptobenzothiazole (MBT) in the presence of non-ionic micelles of Triton X-114. The effect of experimental conditions such as pH, concentration of chelating agent and surfactant, equilibration temperature and time on cloud point extraction was studied. Under the optimum conditions, the preconcentration of 10 mL of water sample in the presence of 0.1% Triton X-114 and 2 × 10⁻⁴ mol L⁻¹ 2-mercaptobenzothiazole permitted the detection of 2.2 ng mL⁻¹ silver. The calibration graph was linear in the range of 10–200 ng mL⁻¹, and the recovery of more than 99% was achieved. The proposed method was used in FAAS determination of Ag(I) in water samples.     

Determination of trace Cd and Pb in natural waters by direct single drop microextraction combined with electrothermal atomic absorption spectrometry

A new method of direct single-drop microextraction combined with electrothermal atomic absorption spectrometry (ETAAS) is presented for the determination of trace Cd and Pb with dithizone (H₂DZ) as chelating reagent. Factors influencing the microextraction efficiency and determination, such as pH, microdrop volume, stirring rate, extraction time were evaluated. Under the optimized experimental conditions, the detection limits of the method are 2 and 90 pg mL⁻¹ for Cd and Pb, and the relative standards deviations for 0.5 ng mL⁻¹ Cd and 10 ng mL⁻¹ Pb are 11 and 12.8%. After 10 min of extraction, the enrichment factors for Cd and Pb are 118 and 90, respectively. The results for the determination of Cd and Pb in tap water, spring water, river water, pond water, lake water and spiked water samples demonstrate the accuracy, recovery and applicability of the method. An environmental water certified reference material (GSBZ 50009-88) was analyzed, and the determined values are in a good agreement with the certified values. Correspondence: Bin Hu, Department of Chemistry, Wuhan University, Wuhan 430072, P.R. China     

Determination of marker pteridines in urine by HPLC with fluorimetric detection and second-order multivariate calibration using MCR-ALS

A liquid chromatographic method has been developed, in combination with the multivariate curve resolution-alternating least squares algorithm (MCR-ALS), for the simultaneous determination of marker pteridines in urine samples. A central composite design has been applied to optimize the factors influencing the separation (buffer concentration, buffer pH, flow rate, oven temperature, mobile-phase composition). A set of 15 calibration samples were randomly prepared, in a concentration range of 0.5–10.5 ng mL⁻¹ for neopterin, biopterin, and pterin; 4.0–8.0 ng mL⁻¹ for xanthopterin; and 0.5–4.5 ng mL⁻¹ for isoxanthopterin. The validation was carried out with fortified urine samples from healthy adults. The optimized conditions were a mobile-phase composition of 10 mM citric buffer at pH 5.44 and acetonitrile (94.5/5.5, v/v), a flow rate of 1.0 mL min⁻¹, and an oven temperature of 25 °C. The detection system consisted of a fast-scanning spectrofluorimeter, which allows obtaining of second-order data matrices containing the fluorescence intensity as a function of retention time and emission wavelength. In this work, MCR-ALS was used to cope with coeluting interferences, on account of the second-order advantage inherent to this algorithm which, in addition, is able to handle data sets deviating from trilinearity, like the high-performance liquid chromatography data analyzed in the present report. The developed approach enabled us to determine five pteridines, some of them with overlapped profiles, reducing the experimental time and reagent consumption. Ratio values for pteridines/creatinine in urine, for infected children with different pathologies, are reported in this work.     

Development of a liquid chromatography-mass spectrometry method for the determination of ursolic acid in rat plasma and tissue: application to the pharmacokinetic and tissue distribution study

A fast and sensitive liquid chromatography–mass spectrometry method was developed for the determination of ursolic acid (UA) in rat plasma and tissues. Glycyrrhetinic acid was used as the internal standard (IS). Chromatographic separation was performed on a 3.5 μm Zorbax SB-C18 column (30 mm × 2.1 mm) with a mobile phase consisting of methanol and aqueous 10 mM ammonium acetate using gradient elution. Quantification was performed by selected ion monitoring with (m/z)⁻ 455 for UA and (m/z)⁻ 469 for the IS. The method was validated in the concentration range of 2.5 − 1470 ng mL⁻¹ for plasma samples and 20 − 11760 ng g⁻¹ for tissue homogenates. The intra- and inter-day assay of precision in plasma and tissues ranged from 1.6% to 7.1% and 3.7% to 9.0%, respectively, and the intra- and inter-day assay accuracy was 84.2 − 106.9% and 82.1 − 108.1%, respectively. Recoveries in plasma and tissues ranged from 83.2% to 106.2%. The limits of detections were 0.5 ng mL⁻¹ or 4.0 ng g⁻¹. The recoveries for all samples were >90%, except for liver, which indicated that ursolic acid may metabolize in liver. The main pharmacokinetic parameters obtained were T _max = 0.42 ± 0.11 h, C _max = 1.10 ± 0.31 μg mL⁻¹, AUC = 1.45 ± 0.21 μg h mL⁻¹ and K _a = 5.64 ± 1.89 h⁻¹. The concentrations of UA in rat lung, spleen, liver, heart, and cerebellum were studied for the first time. This method is validated and could be applicable to the investigation of the pharmacokinetics and tissue distribution of UA in rats.     

In vitro monitoring of clindamycin in human urine using flow injection chemiluminescence

A sensitive chemiluminescence method for the determination of clindamycin is presented. The method is based on the inhibitory effect of clindamycin on the chemiluminescence reaction between luminol and myoglobin in a flow-injection system. The decrement in chemiluminescence intensity is linear with the logarithm of the clindamycin concentration over the range of 0.1–70.0 ng mL⁻¹ (r ² = 0.9995), with a detection limit of 0.03 ng mL⁻¹ (3σ). At a flow rate of 2.0 mL min⁻¹, the complete analytical process could be performed within 0.5 min, including sampling and washing, with a relative standard deviation of less than 3.0% (n = 5). The procedure was applied to the determination of clindamycin in human serum and in monitoring the excretion of clindamycin in human urine samples without any pretreatment process. It was found that the excretive clindamycin concentration reached its maximum 3 hours after oral administration. The clindamycin excretive ratio in 9 hours was 10.84% in the body of the volunteer.     

Analysis of isoflavones and flavonoids in human urine by UHPLC

A rapid, ultra high-performance liquid chromatographic (UHPLC) method has been developed and validated for simultaneous identification and analysis of the isoflavones genistein, daidzein, glycitin, puerarin, and biochanin A, and the flavonoids (±)-catechin, (−)-epicatechin, rutin, hesperidin, neohesperidin, quercitrin, and hesperetin in human urine. Urine samples were incubated with β-glucuronidase/sulfatase. UHPLC was performed with a Hypersil Gold (50 × 2.1 mm, 1.9 μm) analytical column. Elution was with a gradient prepared from aqueous trifluoroacetic acid (0.05%) and acetonitrile. UV detection was performed at 254 and 280 nm. The calibration curves were indicative of good linearity (r ² ≥ 0.9992) in the range of interest for each analyte. LODs ranged between 15.4 and 107.0 ng mL⁻¹ and 3.9 and 20.4 ng mL⁻¹ for flavonoids and isoflavones, respectively. Intra-day and inter-day precision (C.V., %) was less than 3.9% and 3.8%, respectively, and accuracy was between 0.03% and 5.0%. Recovery was 70.35–96.58%. The method is very rapid, simple, and reliable, and suitable for pharmacokinetic analysis. It can be routinely used for simultaneous determination of these five isoflavones and seven flavonoids in human urine. The method can also be applied to studies after administration of pharmaceutical preparations containing isoflavones and flavonoids to humans.     

Electrospun composite of polypyrrole-polyamide as a micro-solid phase extraction sorbent

A micro-solid phase extraction technique was developed using a novel polypyrrole-polyamide nanofiber sheet, fabricated by electrospinning method. The applicability of the new nanofiber sheet was examined as an extracting medium to isolate malathion as a model pesticide from aqueous samples. Solvent desorption was subsequently performed in a microvial, and an aliquot of extractant was injected into gas chromatography–mass spectrometry. Various parameters affecting the electrospinning process including monomer concentration, polyamide content, applied voltage, and electrospinning time were examined. After fabricating the most suitable preparation conditions, influential parameters on the extraction and desorption processes were optimized. The developed method proved to be rather convenient and offers sufficient sensitivity and good reproducibility. The limit of detection (S/N = 3) and limit of quantification (S/N = 10) of the method under optimized conditions were 50 and 100 ng L⁻¹, respectively. The relative standard deviation at concentration level of 1 ng mL⁻¹ was 2% (n = 3). The calibration curve of analyte showed linearity in the range of 0.1–1 ng mL⁻¹ (R ² = 0.9975). The developed method was successfully applied to tap and Zayanderood river water samples, while the relative recovery percentages of 98% and 96% were obtained, respectively. The whole procedure showed to be conveniently applicable and quite easy to be manipulated.     

A spectrofluorimetric method for the determination of acitretin in pharmaceuticals

A simple, rapid, and highly sensitive spectrofluorimetric method for the determination of acitretin was developed based on the strong green fluorescence of acitretin. Influence of organic solvents on the fluorescence spectra of acitretin was studied. Effects of pH, standing time, and foreign ions on the determination of acitretin were also examined. Under the optimum conditions, linear relationship between the relative fluorescence intensity and the concentration of acitretin in the range of 30.0–1100 ng mL⁻¹ was obtained. Detection limit of this method is 9.56 ng mL⁻¹ for acitretin. Relative standard deviation for the determination of 480 ng mL⁻¹ of acitretin was 1.70 %. This method was used for the determination of acitretin in pharmaceuticals and the results were compared with those obtained by the HPLC method.     

Fast screening immunoassay of sulfonamides in commercial fish samples

An indirect competitive enzyme-linked immunosorbent assay (ELISA) was developed in plate to detect three sulfonamide residues (sulfamerazine (SMR), sulfadimetoxine (SDM), and sulfadiazine (SDZ)) in gilthead sea bream (Sparus aurata) samples. Different extraction methodologies—using methanol/water 1:1 (v/v) + ethylene diamine tetraacetic acid (EDTA) 0.5% (m/v), acetonitrile, phosphate-buffered saline (PBS) 10 mmol L⁻¹ pH 7 and acetate buffer 100 mmol L⁻¹ pH 5—and cleanup steps, based on solid-phase extraction (C₁₈, SCX, Si) or liquid extraction with hexane, were assayed. As optimum, a fast and simple method using acetonitrile was selected to extract the sulfonamide residues from the edible muscle of fish. Due to matrix effects, a standard addition calibration curve in fish extract is necessary for quantification purposes. Sulfonamide-free samples were spiked at different concentration levels (between 30 and 90 ng g⁻¹, 5–15 ng mL⁻¹ in plate) and average recoveries (n = 8), ranging from 71% to 95%, 65% to 79%, and 72% to 95%, were obtained for SMR, SDM, and SDZ, respectively. The assay detection limits for these antibiotics were lower than 100 μg kg⁻¹ (maximum residue level established by the European Union). The accuracy was evaluated by spiking blank fish extracts at different concentrations (10–40 ng mL⁻¹, 5–20 ng mL⁻¹ in plate), and the relative errors ranged between ±20%. Finally, in order to confirm the utility of the developed ELISA as a screening methodology, fish samples from different supermarkets were analyzed, and results were compared with those obtained by a validated high-performance liquid chromatography (HPLC) method. The correlation between the results obtained by both ELISA and HPLC methods is satisfactory.      

Dispersive liquid–liquid microextraction followed by gas chromatography–mass spectrometry for the rapid and sensitive determination of UV filters in environmental water samples

The performance of the dispersive liquid–liquid microextraction (DLLME) technique for the determination of eight UV filters and a structurally related personal care species, benzyl salicylate (BzS), in environmental water samples is evaluated. After extraction, analytes were determined by gas chromatography combined with mass spectrometry detection (GC-MS). Parameters potentially affecting the performance of the sample preparation method (sample pH, ionic strength, type and volume of dispersant and extractant solvents) were systematically investigated using both multi- and univariant optimization strategies. Under final working conditions, analytes were extracted from 10 mL water samples by addition of 1 mL of acetone (dispersant) containing 60 μL of chlorobenzene (extractant), without modifying either the pH or the ionic strength of the sample. Limits of quantification (LOQs) between 2 and 14 ng L⁻¹, inter-day variability (evaluated with relative standard deviations, RSDs) from 9% to 14% and good linearity up to concentrations of 10,000 ng L⁻¹ were obtained. Moreover, the efficiency of the extraction was scarcely affected by the type of water sample. With the only exception of 2-ethylhexyl-p-dimethylaminobenzoate (EHPABA), compounds were found in environmental water samples at concentrations between 6 ± 1 ng L⁻¹ and 26 ± 2 ng mL⁻¹.