Comparison of dynamic viscoelastic and physicochemical properties of pressurised and pasteurised longan juices with xanthan addition

Physical and biochemical properties of pressurised and pasteurised longan juices with various xanthan additions, such as viscoelastic behaviour, colour L (lightness), −a^∗ (greenness), b^∗ (yellowness), ΔE (total different colours) and BI (Browning Index) parameters, polyphenol oxidase (PPO) activity, ascorbic acid, gallic acid, ellagic acid, total phenols and antioxidant capacity (DPPH assay) were studied. Viscoelastic determination indicated that longan juice with 0.15% xanthan addition was optimal for a fruit drink. Colour parameters showed pressurised longan juice at 500 MPa was brighter and more transparent than fresh and other processed juices. PPO was completely inactivated in pasteurised juices, whereas in pressurised juices at 300 and 500 MPa, the activities were more than 100% and 95–99%, respectively. Bioactive components including ascorbic acid were significantly reduced according to treatment severities, whereas gallic and ellagic acids were relatively stable in all processed juices. Total phenols and DPPH radical-scavenging activity decreased significantly on pasteurisation, but were stable on pressurisation.     

Development of polyclonal antibody-based indirect competitive enzyme-linked immunosorbent assay for sodium saccharin residue in food samples

A sensitive and specific polyclonal antibody (PcAb)-based indirect competitive enzyme-linked immunosorbent assay (icELISA) for sodium saccharin is described. 6-Amino saccharin was coupled to carrier protein for artificial antigen by diazotisation. New Zealand white rabbits were immunised to obtain anti-sodium saccharin PcAb and then icELISA was developed. The assay showed high sensitivity and specificity to sodium saccharin, with the 50% inhibition value (IC₅₀) of 0.243 μg mL⁻¹, workable range (IC₃₀–IC₇₀) of 0.050–12.8 μg mL⁻¹ and limit of detection (LOD, IC₂₀) of 0.021 μg mL⁻¹. The average recoveries of sodium saccharin in spiked food samples were estimated ranging from 70.7% to 98.8%. A statistically significant correlation of results was obtained between this new ELISA and previously established HPLC approaches with the food-relevant sodium saccharin concentration range 0–320 μg mL⁻¹ (R² = 0.9887–0.9975). These results indicated that the established ELISA was a potential and useful analytical tool for rapid determination of sodium saccharin residue in food samples.     

Industrial orange juice debittering: Impact on bioactive compounds and nutritional value

The impact of an industrial debittering process (DP) on nutritional and bioactive compounds in orange juice (OJ) was studied. The DP was aimed at removing bitter components in OJ by physical adsorption in a resin. The levels of bioactive compounds (carotenoids, ascorbic acid and phenolics), total antioxidant activity and the colour in the fresh orange juices (non-debittered) and in the debittered counterparts were measured. The results demonstrated that the carotenoid contents were not significantly affected by the treatment. However, the debittered orange juices showed a reduction (p < 0.001) of 26% in ascorbic acid, 32% in hydroxycinnamic acids, 28% of flavones and 41% of flavanones in comparison with the non-treated juices. The antioxidant activity of the hydrophilic fraction (HF) was significantly higher (p < 0.05) in untreated juice than in debittered juices. Some colour parameters (L^∗, a^∗ and h_ab) were also affected. Discriminant analysis revealed that the canonical function related to the levels of HF compounds allowed a 100% correct classifications of the different types of juices.     

Physiochemical properties and kinetics of glucoamylase produced from deoxy-d-glucose resistant mutant of Aspergillus niger for soluble starch hydrolysis

Glucoamylases (GAs) from a wild and a deoxy-d-glucose-resistant mutant of a locally isolated Aspergillus niger were purified to apparent homogeneity. The subunit molecular mass estimated by SDS–PAGE was 93 kDa for both strains, while the molecular masses determined by MALDI-TOF for wild and mutant GAs were 72.876 and 72.063 kDa, respectively. The monomeric nature of the enzymes was confirmed through activity staining. Significant improvement was observed in the kinetic properties of the mutant GA relative to the wild type enzyme. Kinetic constants of starch hydrolysis for A. niger parent and mutant GAs calculated on the basis of molecular masses determined through MALDI-TOF were as follows: k_cat = 343 and 727 s⁻¹, K_m = 0.25 and 0.16 mg mL⁻¹, k_cat/K_m (specificity constant) = 1374 and 4510 mg mL⁻¹ s⁻¹, respectively. Thermodynamic parameters for soluble starch hydrolysis also suggested that mutant GA was more efficient compared to the parent enzyme.     

Thermal inactivation of peroxidase during blanching of butternut squash

The kinetics of thermal inactivation of peroxidase in butternut squash during water blanching was studied. Thin slices of squash were immersed in water at 60-90 °C for 0-60 min. For treatments below 70 °C, a biphasic, first-order kinetics model was proposed for peroxidase inactivation that would correspond to heat-labile and heat-resistant fractions of the enzyme. Parameters of the first-order model for the heat-labile and heat-resistant fractions were k_L,ref=24.8733 min⁻¹, E_aL=14023 J/mol and k_R,ref=0.0729 min⁻¹, E_aR=15794 J/mol, respectively. For treatments above 70 °C, a monophasic first-order kinetics model was proposed. Parameters were k_ref=8.6425 min⁻¹, E_a=149900 J/mol. Decreases in ascorbic acid contents due to blanching were analyzed. Blanching processes at high temperature and short time resulted in higher ascorbic acid retention. A linear relationship between ascorbic acid retention and processing temperature was found.     

Nutrient contents of kale (Brassica oleraceae L. var. acephala DC.)

Fructose, glucose and sucrose, as the major soluble sugars and citric and malic acids, as the major organic acids, were identified and determined in kale (Brassica oleraceae L. var. acephala DC., black cabbage) leaves. Fructose was the predominant sugar (2011 mg 100 g⁻¹ dry wt) identified, followed by glucose (1056 mg 100 g⁻¹ dry wt) and sucrose (894 mg 100 g⁻¹ dry wt). The contents of citric and malic acids were at 2213 and 151 mg 100 g⁻¹ dry wt in the leaves. The 16:0, 18:2n − 6 and 18:3n − 3 fatty acids were the most abundant fatty acids in the leaves. Considering the level of these fatty acids, 18:3n − 3 was found to be the highest (85.3 μg g⁻¹ dry wt), contributing 54.0% of the total fatty acid content. Linoleic acid (18:2n − 6), being the second most abundant fatty acid was present at 18.6 μg g⁻¹ dry wt, contributing 11.8% of the total fatty acid content. In the seed oil of kale, 22:1n − 9 was the most abundant fatty acid (4198 μg g⁻¹ dry wt, 45.7%), with 18:2n − 6 (1199 μg g⁻¹ dry wt, 12.3%) and 18:1n − 9 (1408 μg g⁻¹ dry wt, 14.8%) being the second next most abundant fatty acids. The most abundant amino acid was glutamic acid (Glu) which was present at 33.2 mg g⁻¹ dry wt. Aspartic acid, which was the second most abundant amino acid, was present at 27.6 mg g⁻¹ dry wt and accounted for 10.2% of the total amino acid content of kale leaf. The amino acid content was assessed by comparing the percentages of the essential amino acids in kale leaf versus those of a World Health Organization (WHO) standard protein. The protein of kale leaf compares well with that of the WHO standard. Only one amino acid, lysine, had a score that fell below 100%; the lysine score of kale leaf was 95%. This study attempts to contribute to knowledge of the nutritional properties of the plant. These results may be useful for the evaluation of dietary information.     

Hollow fibre-based liquid phase microextraction combined with high-performance liquid chromatography for the analysis of flavonoids in Echinophora platyloba DC. and Mentha piperita

A simple, inexpensive and efficient three phase hollow fibre liquid phase microextraction (HF-LPME) technique combined with HPLC was used for the simultaneous determination of flavonoids in Echinophora platyloba DC. and Mentha piperita. Different factors affecting the HF-LPME procedure were investigated and optimised. The optimised extraction conditions were as follows: 1-octanol as an organic solvent, pH_donor = 2, pH_acceptor = 9.75, stirring rate of 1000 rpm, extraction time of 80 min, without addition of salt. Under these conditions, the enrichment factors ranged between 146 and 311. The values of intra and inter-day relative standard deviations (RSD) were in the range of 3.18–6.00% and 7.25–11.00%, respectively. The limits of detection (LODs) ranged between 0.5 and 7.0 ng mL⁻¹. Among the investigated flavonoids quercetin was found in E. platyloba DC. and luteolin was found in M. piperita. Concentration of quercetin and luteolin was 0.015 and 0.025 mg g⁻¹ respectively.     

Capillary zone electrophoresis of organic acids in beverages

Capillary zone electrophoresis (CZE) with photodiode array detection (PDA) was applied to simultaneously determine eleven organic acids in wine, beer, and fruit and vegetable juices after derivatisation with 2-nitrophenylhydrazine (2-NPH) in the presence of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC·HCl). The parameters affecting CZE separation included type and concentration of buffer, pH, organic additive and applied voltage. Optimum conditions at 25 °C were: 30 mmol l⁻¹ borate buffer, pH 10.0, containing 100 ml l⁻¹ acetonitrile, at 20 kV, sample injection at 0.5 psi for 5 s, with direct detection at 230 nm. Separation of eleven organic acids was achieved within 12 min. Linear calibration curves with good fit were obtained in the range 10.0-100.0 mg l⁻¹. Limits of detection ranged from 2.0 to 10.0 mg l⁻¹. Intra-day precision with RSD?4.0% for migration time and ?5.0% for peak area, and inter-day precision with RSD?6.0% and ?9.0% for migration time and peak area, were obtained. Recovery for all beverage samples was 97±15%.     

Pharmacokinetics and residues of tetracycline in crucian carp muscle using capillary electrophoresis on-line coupled with electrochemiluminescence detection

A novel and sensitive determination for tetracycline (TC) in crucian carp muscle was developed by using capillary electrophoresis coupled with electrochemiluminescence (ECL) detection. The conditions affected separation and detection were examined in detail. The linearity range of TC concentration was from 0.005 to 10 μg mL⁻¹ with a correlation coefficient of 0.9992. The detection limit of TC (S/N = 3) was 1.8 ng mL⁻¹. The relative standard deviations of the ECL intensity and the migration time for eleven consecutive injections of 1.0 μg mL⁻¹ TC were 1.6% and 0.8%, respectively. The recovery of TC was 97.8% (n = 5). After administration of 75 mg kg⁻¹ TC, the maximum concentration and peak time of TC in crucian carp muscle were 7.33 mg kg⁻¹ and 8 h, respectively. After administration of TC, TC concentration demonstrated trivial variation in the period from 48 to 96 h.     

A screening method for the determination of ascorbic acid in fruit juices and soft drinks

A simple and rapid liquid chromatographic method based on a new stationary phase Teknokroma, Tr-010065 Mediterranea sea₁₈ (15 cm × 0.4 cm, id 3 μm), to determine ascorbic acid in beverages is reported. With the proposed method the samples were analysed by direct injection without a previous treatment. The total analysis time does not exceed 6 min. The method showed a good repeatability (RSD < 2%: n = 6) and an excellent sensitivity (LOD = 0.01 mg/l). Seventeen samples were analysed, including fruit juices, soft drinks and isotonic beverages. Ascorbic acid contents ranged from 6.6 to 840 mg/l. The ascorbic acid stability in some beverages during their shelf-life was also evaluated. Degradation of about 54% was observed in a tea drink.     

Determination of flavonoids in propolis-rich functional foods by reversed phase high performance liquid chromatography with diode array detection

A method of reversed phase high performance liquid chromatography (RP-HPLC) coupled with a diode array detector (DAD) was developed for the simultaneous determination of 10 common flavonoids in propolis-rich functional foods. Flavonoids in a sample were extracted with methanol in an ultrasonic bath for 15 min and then chromatographically separated on a C₁₈ column. The binary mobile phase, composed of water with 1% tetrahydrofuran (pH = 2.5) and acetonitrile, was used for gradient elution. The proposed method revealed a good linearity between the peak area of each analyte and its concentration. The intra- and inter-day relative standard deviations (RSDs) were less than 1.90% and 4.91%, respectively. Recoveries were within the range of 84.2–118.7%. LODs and LOQs were 0.043–0.232 μg mL⁻¹ (S/N = 3) and 0.007–0.039 mg g⁻¹ (S/N = 10), respectively. The proposed method provided a simple and rapid routine analysis of common flavonoids in propolis-rich functional foods which are popular on the market nowadays.     

Effect of a commercial adjunct culture on organic acid contents of low-fat Feta-type cheese

The effect of a commercial adjunct culture (CR-213, containing Lactococcus lactis subsp. cremoris and Lactococcus lactis susp. lactis and added at the level of 0.6 g kg⁻¹ or 0.9 g kg⁻¹ cheese milk) on the organic acid (OA) content of low-fat Feta-type cheese was studied. Full-fat (∼220 g kg⁻¹) and a low-fat (∼70 g kg⁻¹) cheeses were used as controls. The main OA of all cheeses throughout ripening were lactic, citric and acetic acids. The effect of ripening time was significant (P < 0.05) for all OA but treatments did not affect acetic, succinic and uric acids. Cheeses with lower fat content were found to contain significantly (P < 0.05) more lactic and citric but less butyric acid than the full-fat control. The addition of the adjunct culture had a positive effect on butyric acid, propionic acid and acetoin content. The use of the adjunct culture could enhance the production of OA in low-fat Feta-type cheeses with eventual positive effect on their sensory properties.     

Efficacy of sodium chlorite as an inhibitor of enzymatic browning in apple slices

Sodium chlorite (SC) is an effective sanitizer for inhibiting microbial growth. This investigation was conducted to determine the efficacy of SC as a browning control agent for use on fresh-cut apple slices, applied alone, or in conjunction with organic acids. Additionally, the authors compared the efficacy of SC to that of acidified sodium chlorite (ASC) and to several other salts and examined the effect of pH and several different organic acids on efficacy of SC. The fresh-cut apple slices were dipped in treatment solutions for 1 min, then drained and placed in plastic containers at 20 °C for 24 h, and finally stored in polyethylene bags at 5 °C for 2 weeks. Color was measured periodically during storage. Lightness (L) values for all treated and control samples measured at 4 h, 24 h, and 2 weeks of storage were compared to L value for untreated samples measured immediately after cutting. Percent decrease in L-values was calculated for each sample at each time interval. Apple slices treated in ASC or SC solution had a significantly smaller decrease in L value indicating less browning than those treated in citric acid or water control at 4 h (P < 0.01), and with the exception of 1 g L⁻¹ ASC and 0.1 g L⁻¹ SC, all other ASC and SC treated slices still had significantly less browning than those for the water control (P < 0.01) at 24 h. After 2 weeks of storage, only SC (0.5–1.0 g L⁻¹), sodium bisulfite (0.5 g L⁻¹) and calcium l-ascorbate (10 g L⁻¹) continued to inhibit browning. Treatment with 0.5 g L⁻¹ SC and pH adjusted in the range from 3.9 to 6.2 using citric acid (CA) reduced browning more effectively than 0.5 g L⁻¹ SC without pH adjustment. Two organic acids, salicylic acid and cinnamic acid, when added to SC solution, were found to achieve even better inhibition of browning than CA at the same pH value.     

Monitoring of fatty acid composition in virgin olive oil by Fourier transformed infrared spectroscopy coupled with partial least squares

A rapid Fourier transformed infrared (FTIR) attenuated total reflectance (ATR) spectroscopic method was applied to the determination of fatty acid (FA) profile and peroxide value (PV) of virgin olive oil. Calibration models were constructed using partial least squares (PLS) regression. A FA calibration model was constructed in the spectral range from 3033 to 700 cm⁻¹. Oleic acid (62.0–80.0%), linoleic acid (5.3–15.0%), saturated fatty acids (SFA, 12.6–19.7%), mono-unsaturated fatty acids (MUFA, 64.4–81.0%) and poly-unsaturated fatty acids (PUFA, 6.0–15.9%) were considered for chemometric analysis. PV (5.7–15.7 meq O₂ kg⁻¹) was calibrated using the signal of the full spectral range 4000–700 cm⁻¹ with first derivative pre-treatment. The LODs of the FTIR-chemometric methods were: 3.0% for oleic acid, 0.5% for linoleic acid, 1.3% for SFA, 3.0% for MUFA, 0.3% for PUFA and 1.0 meq O₂ kg⁻¹ for PV. Analytical methods were evaluated by use of validation samples (n = 25 for all the FA related parameters and n = 10 for PV) with nearly quantitative recovery rates (98–103%). The proposed method provided results comparable to official procedures, with the advantages of being less expensive and more rapid.     

Selective cloud point extraction for the determination of cadmium in food samples by flame atomic absorption spectrometry

A new cloud point extraction (CPE) procedure for preconcentration of cadmium prior to the determination by flame atomic absorption spectrometry (FAAS) was developed. The method is based on the fact that cadmium could form hydrophobic ion-associated complex in the presence of iodide and methyl green (MG), and the hydrophobic ion-associated complex could be extracted into surfactant-rich phase. The main factors affecting CPE procedure, such as pH, concentration of KI, MG and surfactant, equilibrium temperature and incubation time, sample volume were investigated. Potential interference from co-existing ions was largely eliminated as most of co-existing ions can not form extractable ion-associated complex with iodide and MG. Under the optimum conditions, the limit of detection (3σ) and limit of quantity (10σ) were 0.90 ng mL⁻¹ and 3.0 ng mL⁻¹ for cadmium, respectively, and relative standard deviation was 4.2% (c = 50 ng mL⁻¹, n = 7). The proposed method was successfully applied to determination of cadmium in the certified reference rice sample (GBW08510) and food samples with satisfactory results.     

Determination of total arsenic in soft drinks by hydride generation atomic fluorescence spectrometry

A highly sensitive and simple method has been developed for the determination of total arsenic, by continuous hydride generation and atomic fluorescence spectrometry (HGAFS), in refreshing drink samples as colas, teas and fruit juices. Samples were mixed with concentrated HCl and KI to obtain final concentrations of 2 mol l⁻¹ and 1%, respectively. These solutions were aspirated and merged with a reducing NaBH₄ 3% (m/v) solution, with sample and NaBH₄ flow rates of 12.5 and 1.5 ml min⁻¹, respectively. The hydride generated in a 170 cm reaction coil was transported to the detector with an Ar flow of 400 ml min⁻¹. The recovery values of added concentrations, from 0.1 to 0.9 ng ml⁻¹, of arsenic in colas, teas and fruit juices were 94 ± 5, 101 ± 9 and 94 ± 6, respectively, achieving variation coefficients between 0.1% and 9%, confirming the accuracy of developed procedure. Detection limit, ranged from 0.01 to 0.03 ng ml⁻¹. On comparing the direct determination with a reference procedure made after dry ashing operation it can be noticed that the direct approach provides a simplification of the handling and time consuming, achieving statistically comparative results.     

Effect of phosphorus on the fruit yield and food value of two landraces of Trichosanthes cucumerina L.- Cucurbitaceae

Studies were conducted in the early season of 2002 and 2003 at the Teaching and Research Farm, Obafemi Awolowo University, Ile-Ife, Nigeria to evaluate the effect of phosphorus (P) on fruit yield and chemical composition of two landraces of Trichosanthes cucumerina L. For the purpose of the study, two landraces of T. cucumerina named Landrace I and Landrace II were used. The five levels of phosphorus evaluated were 0, 30, 60, 90 and 120 kg P₂O₅ ha⁻¹ using single super phosphate fertilizer (8% P). Statistical analysis showed that 90 kg P₂O₅ ha⁻¹ gave statistically significant higher fruit yield (16.4 tons ha⁻¹) compared to other P levels. The fruit yield of the two Landraces did not differ significantly. Except for crude protein content, the 90 kg P₂O₅ ha⁻¹ produced significantly higher ether extract (1.22 g 100 g⁻¹), crude fibre (1.93 g 100 g⁻¹), moisture content (90.5 g 100 g⁻¹), ash (0.90 g 100 g⁻¹), total sugars (0.81 g 100 g⁻¹) and ascorbic acid (28.7 mg 100 g⁻¹) than other P levels. The essential amino acids compositions were also significantly higher at 90 g 100 g⁻¹ compared to other lower P levels. Landrace I had significantly higher ether extract (0.90 g 100 g⁻¹) content than Landrace II (0.62 g 100 g⁻¹) while Landrace II in turn had significantly higher total sugar (0.76 g 100 g⁻¹) compared to Landrace I (0.61 g 100 g⁻¹). The essential amino acids composition is high and the oxalate composition is low. The high ascorbic acid and amino acid content together with a low oxalate composition suggested a strong basis for encouraging the cultivation of this indigenous fruit vegetable to augment nutrient requirement, improve diet and consequently alleviate poverty, preserve the biodiversity and increase the gene bank of neglected wild species of high quality nutrient sources.     

Estimation of neuroprotective effects of Laurocerasus officinalis Roem. (cherry laurel) by in vitro methods

Dichloromethane, ethyl acetate, acetone, methanol, and water extracts prepared from the fruits and leaves of Laurocerasus officinalis Roem. (LO) (Rosaceae) were screened for their cholinesterase inhibitory activity against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), the key enzymes in pathogenesis of Alzheimer's disease (AD), using ELISA microplate reader at 50, 100, and 200 ??g mL⁻¹. As AD is associated with oxidative stress, the antioxidant activity of the extracts was also tested by radical-forming methods against 2,2-diphenyl-1-picrylhydrazyl (DPPH), N,N-dimethyl-p-phenylendiamine (DMPD), and superoxide radicals as well as iron-related methods; iron-chelating capacity and ferric-reducing antioxidant power (FRAP) assays. Total phenol and flavonoid quantification was achieved using Folin-Ciocalteau and AlCl₃ reagents, respectively. The highest AChE (44.01 ± 1.75%) and BChE (19.91 ± 0.37%) inhibition was caused by the LO-leaf-methanol extract 200 ??g mL⁻¹, while it showed the best radical-scavenging activity against DPPH at 2000 ??g mL⁻¹. Only, the dichloromethane and water extracts of the fruits and the leaf water extract had an iron-chelating capacity, while the leaf methanol extract displayed the highest FRAP. The leaf methanol extract (113.45 ± 0.71 mg g⁻¹ extract) was found to be the richest in total phenols, while the leaf acetone extract (139.90 ± 4.64 mg g⁻¹ extract) had the most abundant amount of total flavonoids.     

Flow injection analysis of total curcuminoids in turmeric and total antioxidant capacity using 2,2′-diphenyl-1-picrylhydrazyl assay

A simple flow injection analysis procedure is proposed for the determination of curcuminoids content in turmeric extracts. The method is based on the formation of a coloured complex between 4-aminoantipyrine and curcuminoids, in the presence of an oxidising reagent such as potassium hexacyanoferrate (III) in alkaline media. Conditions selected as a result of these trials were implemented in a flow injection analytical system in which the influence of injection volume, flow rate, reagent concentration and mixing coil length, was evaluated. Under the optimum conditions the total amount of curcuminoids could be determined within a concentration range of 5–50 μg mL⁻¹ which can be expressed by the regression equation y = 0.003x − 0.0053 (r² = 0.9997). The limits of detection and quantitation were found to be 0.6 μg mL⁻¹ and 1.8 μg mL⁻¹, respectively. The reproducibility of analytical readings was indicative of standard deviations <2%. The sample was extracted and analysed by using the proposed method. The percentage recoveries were found to be 94.3–108.0. The proposed system was applied to the determination of curcuminoids content in turmeric. The total curcuminoid contents in turmeric extract were found to be 0.9–4.3% (w/w). The development method is simple, economic, rapid and especially suitable for quality control in pharmaceutical plants.     

Analysis of multi-pesticide residues in the foods of animal origin by GC–MS coupled with accelerated solvent extraction and gel permeation chromatography cleanup

A new analytical method was developed to simultaneously determine residues of 109 pesticides (including isomers) in the foods of animal origin. Acetonitrile was selected for accelerated solvent extraction (ASE) for effectively extracting the pesticides from the fatty samples. The cleanup was performed with an automated gel permeation chromatography (GPC) cleanup system. The prepared samples were analysed with GC–MS in the selected ion monitoring mode (SIM) using one target and two qualitative ions for each analyte. Chlorpyrifos-d₁₀ was used as an internal standard. The lowest limit of detection was 0.3 μg kg⁻¹ for some pesticides. The recoveries and relative standard deviations (RSDs) were checked by spiking untreated samples with pesticides at 0.05, 0.1 and 0.2 mg kg⁻¹. The average recoveries of most pesticides were from 62.6% to 107.8%. The precision values expressed as RSD were all ?20.5% (n = 6). Good linearity (r ? 0.99) was observed between 0.05 and 1.5 μg mL⁻¹.