Pretreatment with dilute maleic acid enhances the enzymatic digestibility of sugarcane bagasse and oil palm empty fruit bunch fiber.

Lignocellulose is resistant to degradation and requires pretreatment before hydrolytic enzymes can release fermentable sugars. Sulfuric acid has been widely used for biomass pretreatment, but high amount of degradation products usually occurred when using this method. To enhance accessibility to cellulose, we studied the performances of several dilute organic acid pretreatments of sugarcane bagasse and oil palm empty fruit bunch fiber. The results revealed that pretreatment with maleic acid yields the highest xylose and glucose release among other organic acids. The effects of concentration, duration of heating and heating temperature were further studied. Dilute maleic acid 1 % (w/w) pretreatment at 180 °C was the key to its viability as a substitute for sulfuric acid. Moreover, maleic acid did not seem to highly promote the formation of either furfural or 5-HMF in the liquid hydrolysate after pretreatment.

Determining the effect of ocular chemical injuries on topical drug delivery.

Ocular chemical injuries (OCIs) commonly cause ocular damage and visual loss and treatment uses topical therapies to facilitate healing and limit complications. However, the impact of chemical injury on corneal barrier function and treatment penetration is unknown. Therefore, the aim of this study was to determine the effect of OCI on drug penetration and absorption. Porcine corneal explants were used to assess histological damage, electrical resistance, and the trans-corneal penetration/corneal adsorption of reference compounds (sodium fluorescein and rhodamine B) and dexamethasone. Corneal explants were injured with either 1 M sulfuric acid, or 1 M sodium hydroxide. Dexamethasone penetration was measured using high-performance liquid chromatography (HPLC) and that of fluorescein and rhodamine using fluorescence. Dexamethasone corneal adsorption was measured using enzyme-linked immunoabsorbant assay (ELISA). Both acid and alkaline injuries reduced trans-corneal electrical resistance. NaOH injury increased hydrophilic fluorescein penetration (NaOH 8.59 ± 1.50E-05 cm.min-1 vs. Hanks' Balanced Salt Solution (HBSS) 1.64 ± 1.01E-06 cm.min-1) with little impact on hydrophobic rhodamine B (1 M NaOH 6.55 ± 2.45E-04 cm.min-1 vs. HBSS 4.60 ± 0.972E-04 cm.min-1) and dexamethasone penetration (1 M NaOH 3.00 ± 0.853E-04 cm.min-1 vs. HBSS 2.69 ± 0.439E-04 cm.min-1). By contrast, H2SO4 decreased trans-corneal penetration of hydrophilic fluorescein (H2SO4 1.16 ± 14.2E-07 cm.min-1) and of hydrophobic dexamethasone (H2SO4 1.88 ± 0.646E-04 cm.min-1) and rhodamine B (H2SO4 4.60 ± 1.42E-05 cm.min-1). Acid and alkaline OCI differentially disrupted the corneal epithelial barrier function. Acid injury reduced penetration of hydrophobic dexamethasone and rhodamine B as well as hydrophilic fluorescein, which may translate clinically into reduced drug penetration after OCI, while alkaline injury increased fluorescein penetration, with minimal effect on dexamethasone and rhodamine B penetration.

Insights into waxy maize starch degradation by sulfuric acid: Impact on starch structure, pasting, and rheological property.

This study investigated the effects of acid degradation of amylopectin on the structure, pasting, and rheological properties of waxy maize starch. It is found that: 1) the amount of amylopectin short-chains with degree of polymerization (DP) ~ 15-50 increased while that of amylopectin long-chains with DP ~ 50-200 decreased by acid hydrolysis; 2) acid hydrolysis produced smaller amylopectin molecules with a narrower size distribution; 3) acid hydrolysis had a minor effect on the crystalline and granular structures of native starch; 4) the pasting viscosity of acid hydrolyzed starch during heating and the consistency coefficient, K, of starch gels increased, whereas the flow behavior index, n, decreased. Correlation analysis was used to clarify the molecular causes for the variations of pasting and rheological properties of acid hydrolyzed starch.

Anti-Acanthamoeba disinfection: hands, surfaces and wounds.

Acanthamoebae are facultative parasites causing rare but serious infections such as keratitis and encephalitis and are also known as vectors for several bacterial pathogens, including legionellae and pseudomonads. Acanthamoeba cysts are particularly resilient and enable the amoebae to withstand desiccation and to resist disinfection and therapy. While the search for new therapeutic options has been intensified in the past years, hand and surface disinfectants as well as topical antiseptics for preventing infections have not been studied in detail to date. The aim of this study was to screen well-known and commonly used antimicrobial products in various formulations and different concentrations for their efficacy against Acanthamoeba trophozoites and cysts, including aliphatic alcohols, quaternary ammonium compounds (QACs), peracetic acid (PAA), potassium peroxymonosulfate sulfate (PPMS) and octenidine dihydrochloride (OCT). Of all products tested, OCT and QACs showed the highest efficacy, totally eradicating both trophozoites and cysts within 1 min. The determined 50% effective concentration (EC50) for cysts was 0.196 mg/mL for OCT and 0.119 mg/mL for QACs after 1 min of exposure. PAA and PPMS showed reliable cysticidal efficacies only with prolonged incubation times of 30 min and 60 min, respectively. Aliphatic alcohols generally had limited efficacy, and only against trophozoites. In conclusion, OCT and QACs are potent actives against Acanthamoeba trophozoites and cysts at concentrations used in commercially available products, within contact times suitable for surface and hand disinfection as well as topical antisepsis.

Designing Efficient Processes for Sustainable Bioethanol and Bio-Hydrogen Production from Grass Lawn Waste.

The effect of thermal, acid and alkali pretreatment methods on biological hydrogen (BHP) and bioethanol production (BP) from grass lawn (GL) waste was investigated, under different process schemes. BHP from the whole pretreatment slurry of GL was performed through mixed microbial cultures in simultaneous saccharification and fermentation (SSF) mode, while BP was carried out through the C5yeast Pichia stipitis, in SSF mode. From these experiments, the best pretreatment conditions were determined and the efficiencies for each process were assessed and compared, when using either the whole pretreatment slurry or the separated fractions (solid and liquid), the separate hydrolysis and fermentation (SHF) or SSF mode, and especially for BP, the use of other yeasts such as Pachysolen tannophilus or Saccharomyces cerevisiae. The experimental results showed that pretreatment with 10 gH2SO4/100 g total solids (TS) was the optimum for both BHP and BP. Separation of solid and liquid pretreated fractions led to the highest BHP (270.1 mL H2/g TS, corresponding to 3.4 MJ/kg TS) and also BP (108.8 mg ethanol/g TS, corresponding to 2.9 MJ/kg TS) yields. The latter was achieved by using P. stipitis for the fermentation of the hydrolysate and S. serevisiae for the solid fraction fermentation, at SSF.

Effective microorganisms inoculant: Diversity and effect on the germination of palisade grass seeds.

Effective microorganisms (EM) are inoculants formed by fungi and bacteria isolated from soil. EM are commonly used by farmers on agronomic crops to stimulate plant growth, but their composition and their benefits has been controverted. This study aimed to analyze the diversity of microorganisms growing in three EM inoculants, as well as to evaluate their efficiency in the germination of palisade grass seeds. The total DNA of the three EM inoculants was extracted, the 16S rRNA and ITS genes were amplified by PCR and sequenced on the Illumina MiSeq platform. Germination tests were conducted with three type of the EM, in three concentration and two times of the immersion. The bacterial group was the most abundant in EM, followed by fungi. Bacterial operational taxonomic units OTUs were shared by all EMs. Pre-treatments of palisade grass seeds with EMs resulted in a higher germination percentage (% G) and germination speed index (IVG) when EM was used at concentration of 1 or 2% in water. Seed immersion for 5 min was more efficient than immersion for 24 h. We can conclude that EM of different origin can share microbial groups and diversity of microorganisms, besides being an alternative to increase palisade grass seeds germination.

Biogas Production from Physicochemically Pretreated Grass Lawn Waste: Comparison of Different Process Schemes.

Various pretreatment methods, such as thermal, alkaline and acid, were applied on grass lawn (GL) waste and the effect of each pretreatment method on the Biochemical Methane Potential was evaluated for two options, namely using the whole slurry resulting from pretreatment or the separate solid and liquid fractions obtained. In addition, the effect of each pretreatment on carbohydrate solubilization and lignocellulossic content fractionation (to cellulose, hemicellulose, lignin) was also evaluated. The experimental results showed that the methane yield was enhanced with alkaline pretreatment and, the higher the NaOH concentration (20 g/100 gTotal Solids (TS)), the higher was the methane yield observed (427.07 L CH4/kg Volatile Solids (VS), which was almost 25.7% higher than the BMP of the untreated GL). Comparing the BMP obtained under the two options, i.e., that of the whole pretreatment slurry with the sum of the BMPs of both fractions, it was found that direct anaerobic digestion without separation of the pretreated biomass was favored, in almost all cases. A preliminary energy balance and economic assessment indicated that the process could be sustainable, leading to a positive net heat energy only when using a more concentrated pretreated slurry (i.e., 20% organic loading), or when applying NaOH pretreatment at a lower chemical loading.

The alkylaminoalkanethiosulfuric acids exhibit in-vitro antileishmanial activity against Leishmania (Viannia) braziliensis: a new perspective for use of these schistosomicidal agents.

The alkylaminoalkanethiosulfuric acids (AAATs) are amphipathic compounds effective against experimental schistosomiasis, of low toxicity, elevated bioavailability after a single oral dose and prompt tissue absorption. OBJECTIVES: To explore the in-vitro antileishmanial potential of AAATs using five compounds of this series against Leishmania (Viannia) braziliensis. METHODS: Their effects on promastigotes and axenic amastigotes, and cytotoxicity to macrophages were tested by the MTT method, and on Leishmania-infected macrophages by Giemsa stain. Effects on the mitochondrial membrane potential of promastigotes and axenic amastigotes and DNA of intracellular amastigotes were tested using JC-1 and TUNEL assays, respectively. KEY FINDINGS: The 2-(isopropylamino)-1-octanethiosulfuric acid (I) and 2-(sec-butylamino)-1-octanethiosulfuric acid (II) exhibit activity against both promastigotes and intracellular amastigotes (IC50 25-35 µm), being more toxic to intracellular parasites than to the host cell. Compound I induced a loss of viability of axenic amastigotes, significantly reduced (30%) the mitochondrial membrane potential of both promastigotes and axenic amastigotes and promoted selective DNA fragmentation of the nucleus and kinetoplast of intracellular amastigotes. CONCLUSIONS: In this previously unpublished study of trypanosomatids, it is shown that AAATs could also exhibit selective antileishmanial activity, a new possibility to be investigated in oral treatment of leishmaniasis.

Simple method for analyzing the purity of protease-containing samples by acid-treatment SDS-PAGE.

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is a widely used technique to analyze the purity of a protein. However, it is necessary to denature (via boiling) the samples before subjecting them to electrophoresis. In the case of protease-containing samples, autolysis of the protease can occur, affecting the accuracy of results. In this study, we investigated the methods for analyzing the purity of Dispase I, a thermolysin-like neutral protease. When we analyzed D protease, a neutral metalloprotease component of Dispase I and highly purified Dispase I using the conventional SDS-PAGE method, a large number of bands were detected in both cases. These bands (putative D protease fragments) were assumed to result from autolysis. To inactivate D protease (optimal pH 7-8), 0.05 M sulfuric acid was utilized (pH 0.7-2.5). Using a conventional sample preparation solution, acid-treated Dispase I samples (without boiling) were made, and SDS-PAGE (15% w/v gel) was carried out. Our findings show that autolysis was inhibited under strong acidic conditions, and protein denaturation was achieved by treatment with sulfuric acid and SDS without boiling. Using this modified SDS-PAGE method, the purities of Dispase I and the purified enzyme were determined to be approximately 80% and 98%, respectively. Furthermore, we demonstrated that this method can be applied for the analysis of other samples including non-acidic proteases (e.g., thermolysin, subtilisin, and trypsin) and protease-contaminated samples (a mixed solution of albumin and D protease).

Effects of acid-alkali treatment on bioactivity and osteoinduction of porous titanium: An in vitro study.

BACKGROUND: To elucidate the bioactivity and bone regeneration of porous titanium surfaces treated using acid-alkali combination, and to define the optimal alkali reaction time. METHODS: Ten groups of porous Ti with at least 3 per group undergoing different acid-alkali treated time were prepared. The surface was characterized by scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDS), bicinchoninic acid method (BCA), optical contact angle measurement and Raman spectrometry. Compression testing was performed with a universal testing machine. The bioactivity and osteoinduction were evaluated by a series of biological tests using a simulated body fluid (SBF) test, cell proliferation test, vinculin, ALP and OCN expression, and cell mineralization. RESULTS: The acid-alkali treatment formed micro- and nano-scale structures on the sample surfaces. The alkali treatment for 12 h achieved the sharpest nano-scale surface relief and the most protein absorption. The treated porous surface was coated with a NaHTiO3 layer. The acid-alkali etching did not compromise the elastic modulus and compressive strength of the porous Ti samples. In addition to hydroxyapatite, a perovskite phase was also formed on the treated porous samples in SBF. Non-treated dense Ti showed more cell adhesion and proliferation (P < 0.05), while osteoinduction and mineralization were more pronounced on the treated porous sample (P < 0.05). CONCLUSION: Acid-alkali treatment is an effective means of generating nano-scale relief on porous Ti surface, and is beneficial for bioactivity and bone regeneration. The 15 min acid and 12 h alkali etching is the optimal combination. The osteoinductive efficacy may be attributable to the surface physical chemistry and the formation of hydroxyapatite and perovskite layers, rather than direct cell adhesion and proliferation.

Oxidation of 5-methylaminomethyl uridine (mnm5U) by Oxone Leads to Aldonitrone Derivatives.

Oxidative RNA damage is linked to cell dysfunction and diseases. The present work focuses on the in vitro oxidation of 5-methylaminomethyl uridine (mnm5U), which belongs to the numerous post-transcriptional modifications that are found in tRNA. The reaction of oxone with mnm5U in water at pH 7.5 leads to two aldonitrone derivatives. They form by two oxidation steps and one dehydration step. Therefore, the potential oxidation products of mnm5U in vivo may not be only aldonitrones, but also hydroxylamine and imine derivatives (which may be chemically more reactive). Irradiation of aldonitrone leads to unstable oxaziridine derivatives that are susceptible to isomerization to amide or to hydrolysis to aldehyde derivative.

Pre-treatment of corn stover, Cynara cardunculus L. stems and wheat straw by ethanol-water and diluted sulfuric acid: Comparison under different energy input conditions.

Ethanol-water (EW) and diluted sulfuric acid (DSA) pre-treatment have been studied for lignocellulosic biomass (corn stover, Cynara cardunculus L. stems and wheat straw). Both pre-treatments have been compared taken into account: solids recovery, glucans recovery, xylans removed, delignification and glucose yield. In all cases, the amount of energy involved has been taken as a criterion for sustainability. In general terms, EW is more efficient to remove lignin and DSA more appropriate to hydrolysate xylans. The combined effect of delignification and xylans removal is responsible for the improvement in the enzymatic cellulose hydrolysis. Under conditions of moderate-low energy inputs, EW pre-treatment yields better results than DSA with glucose yields in the range of 50-60% for EW pre-treated corn stover and cardoon stems; while wheat straw pulps reach up to 80%. So, multiple raw materials biorefinery needs a previous study to fit the type and conditions of the pre-treatment to each feedstock.

Smart Card Decontamination in a High-Containment Laboratory.

Validated procedures for decontamination of laboratory surfaces and equipment are essential to biosafety and biorisk programs at high-containment laboratories. Each high-containment laboratory contains a unique combination of surfaces, procedures, and biological agents that require decontamination methods tailored to specific facility practices. The Plum Island Animal Disease Center (PIADC) is a high-containment laboratory operating multiple biosafety level (BSL)-3, ABSL-3, and BSL-3 Ag spaces. The PIADC facility requires the use of federally issued smart cards, called personal identity verification (PIV) cards, to access information technology (IT) networks both outside and within the high-containment laboratory. Because PIV cards may require transit from the BSL-3 to office spaces, a validated procedure for disinfecting PIV card surfaces prior to removal from the laboratory is critical to ensure biosafety and biosecurity. Two high-risk select agents used in the PIADC high-containment laboratory are foot-and-mouth disease virus (FMDV) and swine vesicular disease virus (SVDV). We evaluated disinfection of PIV cards intentionally spotted with FMDV and SVDV using a modified quantitative carrier test and the liquid chemical disinfectant Virkon® S. Our experimental design modeled a worst-case scenario of PIV card contamination and disinfection by combining high concentrations of virus dried with an organic soil load and use of aged Virkon® S prepared in hard water. Results showed that FMDV and SVDV dried on PIV card surfaces were completely inactivated after immersion for 30 and 60 seconds, respectively, in a 5-day-old solution of 1% Virkon® S. Therefore, this study provided internal validation of PIADC biosafety protocols by demonstrating the efficacy of Virkon® S to inactivate viruses on contaminated smart cards at short contact times.

Aryl fluorosulfate analogues as potent antimicrobial agents: SAR, cytotoxicity and docking studies.

A series of aryl fluorosulfate analogues (1-37) were synthesized and tested for in vitro antibacterial and antifungal studies, and validated by docking studies. The compounds 9, 12, 14, 19, 25, 26, 35, 36 and 37 exhibited superior antibacterial potency against tested bacterial strains, while compounds 2, 4, 5, 15, 35, 36 and 37 were found to have better antifungal activity against tested fungal strains, compared to standard antibiotic gentamicin and ketoconazole respectively. Among all the synthesized 37 analogs, compounds 25, 26, 35, 36 and 37 displayed excellent anti-biofilm property against Staphylococcus aureus. The structure-activity relationship (SAR) revealed that the antimicrobial activity depends upon the presence of -OSO2F group and slender effect of different substituent's on the phenyl rings. The electron donating (OCH3) groups in analogs increase the antibacterial activity, and interestingly the electron withdrawing (Cl, NO2, F and Br) groups increase the antifungal activity (except compound 35, 36 and 37). The mechanism of potent compounds showed membrane damage on bacteria confirmed by SEM. Compounds 35, 36 and 37 exhibited highest glide g-scores in molecular docking studies and validated the biocidal property.

FTIR as an easy and fast analytical approach to follow up microbial growth during fungal pretreatment of poplar wood with Phanerochaete chrysosporium.

Since the determination of the fermentation kinetics is one of the main challenges in solid state fermentation, the quantitative measurement of biomass growth during microbial pretreatment by FTIR spectroscopy in Attenuated Total Reflectance mode was evaluated. Peaks at wave numbers of 1651 cm-1 and 1593 cm-1 showed to be affected during pretreatment of poplar wood particles by Phanerochaete chrysosporium MUCL 19343. Samples with different microbial biomass fractions were obtained from two different experiments, i.e., shake flask and fixed-bed reactor experiments. The glucosamine concentration was compared to the normalized absorbance ratio of the 1651 cm-1 to 1593 cm-1 peak, measured by FTIR-ATR, and resulted in a linear relationship. The application of a normalized absorbance ratio in function of time provided a graph that was similar to the microbial growth curve. Application of FTIR in ATR mode to follow-up kinetics during solid state fermentation seems to be a fast and easy alternative to laborious measurement techniques, such as glucosamine determination.

In vitro study of disinfectants on the embryonation and survival of Toxascaris leonina eggs.

The effect of six available and commercial disinfectants on the embryonation and larval development of Toxascaris leonina eggs was studied. Dettol® and Virkon® both induced a 100% reduction in larval development (P ≤ 0.05). Dettol® resulted in deformed eggshells and a halt in embryonal development at 1 week post exposure. All Virkon®-treated eggs showed an early embryonic lysis 24 h post exposure. TH4+ and 70% ethanol both significantly (P ≤ 0.05) affected larval development, with 58.8 and 85.8% reduction, respectively. Neither sodium hypochlorite nor phenol significantly affected larval development (2.8 and 21.0%, respectively). Sodium hypochlorite treatment caused a visible decortication of the eggshell; however, phenol-treated embryonated Toxascaris eggs appeared more or less morphologically normal. In conclusion, the disinfectants tested induced variable degrees of decortication and suppression of larval development. Virkon®S was the most effective disinfectant against Toxascaris eggs, suggesting that it is the most advisable one to use. To the best of our knowledge, this is the first report of the use of Virkon®S as an ovicide and/or larvicide of helminths, particularly Toxascaris leonina.

Production of low-molecular weight soluble yeast ß-glucan by an acid degradation method.

ß-glucan is widely distributed in nature as water soluble and insoluble forms. Both forms of ß-glucan are utilized in several fields, especially for functional foods. Yeast ß-glucan is a medically important insoluble particle. Solubilization of yeast ß-glucan may be valuable for improving functional foods and in medicinal industries. In the present study, we applied an acid degradation method to solubilize yeast ß-glucan and found that ß-glucan was effectively solubilized to low-molecular weight ß-glucans by 45% sulfuric acid treatment at 20°C. The acid-degraded soluble yeast ß-glucan (ad-sBBG) was further fractionated into a higher-molecular weight fraction (ad-sBBG-high) and a lower-molecular weight fraction (ad-sBBG-low). Since ad-sBBG-high contained mannan, while ad-sBBG-low contained it only scarcely, it was possible to prepare low-molecular weight soluble ß-glucan with higher purity. In addition, ad-sBBG-low bound to dectin-1, which is an innate immunity receptor of ß-glucan, and showed antagonistic activity against reactive oxygen production and cytokine synthesis by macrophages. Thus, this acid degradation method is an important procedure for generating immune-modulating, low-molecular weight, soluble yeast ß-glucan.

Efficacy of a Blend of Sulfuric Acid and Sodium Sulfate against Shiga Toxin-Producing Escherichia coli, Salmonella, and Nonpathogenic Escherichia coli Biotype I on Inoculated Prerigor Beef Surface Tissue.

A study was conducted to investigate the efficacy of a sulfuric acid-sodium sulfate blend (SSS) against Escherichia coli O157:H7, non-O157 Shiga toxin-producing E. coli (STEC), Salmonella, and nonpathogenic E. coli biotype I on prerigor beef surface tissue. The suitability of using the nonpathogenic E. coli as a surrogate for in-plant validation studies was also determined by comparing the data obtained for the nonpathogenic inoculum with those for the pathogenic inocula. Prerigor beef tissue samples (10 by 10 cm) were inoculated (ca. 6 log CFU/cm2) on the adipose side in a laboratory-scale spray cabinet with multistrain mixtures of E. coli O157:H7 (5 strains), non-O157 STEC (12 strains), Salmonella (6 strains), or E. coli biotype I (5 strains). Treatment parameters evaluated were two SSS pH values (1.5 and 1.0) and two spray application pressures (13 and 22 lb/in2). Untreated inoculated beef tissue samples served as controls for initial bacterial populations. Overall, the SSS treatments lowered inoculated (6.1 to 6.4 log CFU/cm2) bacterial populations by 0.6 to 1.5 log CFU/cm2 (P < 0.05), depending on inoculum type and recovery medium. There were no main effects (P ≥ 0.05) of solution pH or spray application pressure when SSS was applied to samples inoculated with any of the tested E. coli inocula; however, solution pH did have a significant effect (P < 0.05) when SSS was applied to samples inoculated with Salmonella. Results indicated that the response of the nonpathogenic E. coli inoculum to the SSS treatments was similar (P ≥ 0.05) to that of the pathogenic inocula tested, making the E. coli biotype I strains viable surrogate organisms for in-plant validation of SSS efficacy on beef. The application of SSS at the tested parameters to prerigor beef surface tissue may be an effective intervention for controlling pathogens in a commercial beef harvest process.

Graphite-Based Nanocomposite Electrochemical Sensor for Multiplex Detection of Adenine, Guanine, Thymine, and Cytosine: A Biomedical Prospect for Studying DNA Damage.

Guanine (G), adenine (A), thymine (T), and cytosine (C) are the four basic constituents of DNA. Studies on DNA composition have focused especially on DNA damage and genotoxicity. However, the development of a rapid, simple, and multiplex method for the simultaneous measurement of the four DNA bases remains a challenge. In this study, we describe a graphite-based nanocomposite electrode (Au-rGO/MWCNT/graphite) that uses a simple electro-co-deposition approach. We successfully applied the developed sensor for multiplex detection of G, A, T, and C, using square-wave voltammetry. The sensor was tested using real animal and plant DNA samples in which the hydrolysis of T and C could be achieved with 8 mol L-1 of acid. The electrochemical sensor exhibited excellent sensitivity (G = 178.8 nA/µg mL-1, A = 92.9 nA/µg mL-1, T = 1.4 nA/µg mL-1, and C = 15.1 9 nA/µg mL-1), low limit of detection (G, A = 0.5 µg mL-1; T, C = 1.0 µg mL-1), and high selectivity in the presence of common interfering factors from biological matrixes. The reliability of the established method was assessed by method validation and comparison with the ultraperformance liquid chromatography technique, and a correlation of 103.7% was achieved.