Analytical Methods (v.10, #16)

Front cover (1791-1792).

Contents list (1793-1798).

Antimicrobial susceptibility testing by using virulent phages to evaluate bacterial viability by Yong He; Enci Fan; Mengyao Wang; Shuguang Lu; Zhifeng Fu (1799-1804).
One of the most efficient approaches for minimizing antimicrobial resistance of pathogenic bacteria is to develop rapid and reliable diagnostic protocols for antimicrobial susceptibility testing (AST). Here a multidrug resistant Pseudomonas aeruginosa (P. aeruginosa) strain was used to isolate its virulent phages. Then the virulent phages were adopted to establish a bioluminescent (BL) method for evaluating bacterial viability. Since the viability of susceptible bacteria could be inhibited or eliminated by antibiotics, an AST protocol was designed by utilizing virulent phages to evaluate the viability of antibiotics-exposed bacteria. Minimum bactericidal concentrations (MBCs) could be easily obtained through comparing the BL signals of antibiotics-exposed bacteria in the presence and absence of phages. Within 3 h, the MBCs of tobramycin, gentamicin, ceftazidime, piperacillin and levofloxacin were evaluated to be <4 μg mL−1, 8 μg mL−1, >32 μg mL−1, >128 μg mL−1 and >8 μg mL−1, respectively. The protocol has immense potential to reduce the frequency of empirical antibiotic therapy. It can be extended to the AST of other pathogens by isolation of their virulent phages.

The erythrocyte sedimentation rate (ESR) and red blood cell (RBC) aggregation have been widely used to detect inflammatory diseases. In this study, a simple measurement method is proposed to simultaneously quantify RBC aggregation and the ESR in a driving syringe. A microfluidic device composed of inlets, outlets, and identical parallel channels (counter-fluid channel and blood channel) is developed. The counter-fluid channel and the blood channel are filled with a 40% glycerin solution and blood, respectively. To control counter-fluid flow, a pinch valve is installed at a specific position along the length of the tube connected to the counter-fluid channel. Blood in the driving syringe (Vblood = 0.7–1 mL, hematocrit = 30%) is delivered to the microfluidic device at a pulse-shaped flow rate (Q0 = 1 mL h−1, T = 240 s). As blood pressure is proportional to the volume of RBCs in the counter-fluid channel, blood pressure is measured as Ipress by quantifying the volume of RBCs in the counter-fluid channel. The ESR in the driving syringe is then measured as Ipress or the time constant of Ipress. RBC aggregation is evaluated as IRA by quantifying the image intensity of blood in the blood channel. Blood is first prepared by adding normal RBCs to a dextran solution (Cdextran = 0–10 mg mL−1). The ESR and RBC aggregation in the driving syringe significantly increase at higher dextran concentrations. Second, the proposed method is applied to detect heterogeneous blood samples that are prepared by mixing RBC-aggregated blood with RBC-hardened blood, partially (i.e., mixing ratio = 0, 0.5, and 1). Then, three different blood samples are completely separated with the two parameters (Ipress and IRA). In conclusion, the proposed method can be used to measure RBC aggregation and the ESR in a driving syringe by quantifying the blood pressure index and the RBC aggregation index.

Adsorption and HPLC of carbohydrates and related hydroxy compounds on zeolites by Wolfgang Wach; Iris Fornefett; Christoph Buttersack; Klaus Buchholz (1817-1832).
Carbohydrates, sugar alcohols, and other hydroxy compounds were separated on a HPLC-column filled with dealuminated hydrophobic Y zeolite with water as eluent. Disaccharides show a greater retention and selectivity than monosaccharides. The selectivity for monosaccharides is in the same range compared with established stationary phases but significantly greater for disaccharides and trisaccharides. When a usual hydrophilic Y zeolite is used, either exchanged by K+ or Ca++, all disaccharides are excluded from the micropores and only monosaccharides can be separated. A hydrophobic MOR type zeolite with somewhat smaller pore geometry also excludes the disaccharides while an acceptable selectivity was observed for monosaccharides and other small hydroxyl compounds. The separation can be limited by diffusion of the solutes inside the micropores. Therefore, the separation can be improved by enhancing the temperature. The linear correlation of the dynamic separation process with the static adsorption equilibrium measured after 24 h is limited to low solute–zeolite interactions.

A highly selective “turn-on” fluorescent sensor for zinc ion based on a cinnamyl pyrazoline derivative and its imaging in live cells by Yun-Shang Yang; Chun-Mei Ma; Ying-Peng Zhang; Qing-Hua Xue; Jia-Xi Ru; Xiao-Yu Liu; Hui-Chen Guo (1833-1841).
A fluorescence probe based on cinnamyl pyrazoline was developed and synthesized. The probe detected Zn2+ ion that was mixed with a series of cations in aqueous ethanol with high selectivity and high sensitivity. After adding Zn2+, the fluorescence intensity was significantly enhanced, with a 56-fold increase. The detection limits and binding constants of 2.95 × 10−8 M and 1.468 × 104 M−1 were calculated by a fluorescence titration experiment. The 1 : 1 binding chemometrics of the 1–Zn2+ complex was analyzed by Job's plot. The probe showed good reversibility when adding PPi. Additionally, the “turn-on” fluorescent probe with excellent membrane permeability was satisfactorily applied to microscopic imaging for the detection of Zn2+ in baby hamster kidney (BHK)-21 cells.

Development and validation of a stability-indicating ultra-performance liquid chromatography (UPLC) method for doxycycline hyclate: an optimization of the analytical methodology for a medical countermeasure (MCM) drug by Adil Mohammad; Cheng H. Yen; Miah Schneider; Bryan Lowry; Firat Yerlikaya; Gretchen Whitesell; Brad Leisssa; Patrick J. Faustino; Saeed R. Khan (1842-1851).
Doxycycline hyclate (DOX) is a broad-spectrum anti-infective drug that can be administered for post-exposure prophylaxis (PEP) following exposure to aerosolized spores of Bacillus anthracis and it may be used for acute radiation syndrome (ARS). Because of this public health importance, several federal, state, and local public health and emergency response stakeholders stockpile doxycycline tablets or capsules as medical countermeasures (MCMs). A robust and efficient ultra-performance liquid chromatography (UPLC) method was developed and validated for the simultaneous determination and quantification of doxycycline hyclate and its impurities to enhance the efficiency of control strategies over the product lifecycle. The chromatography was performed on a Waters UPLC system using an Acquity BEH C18 (2.1 × 50 mm, 1.7 μm) column maintained at 55 °C. Doxycycline and its degradation and synthetic impurities were eluted isocratically with 75 mM ammonium acetate, 4 mM EDTA (pH 8.8) and acetonitrile (97 : 3) as the mobile phase with a flow rate of 1.2 mL min−1 and ultraviolet detection at 270 nm. Baseline separation of the analytes was achieved in 6 min. The UPLC method was demonstrated to be stability-indicating after forced degradation studies were conducted on doxycycline hyclate under standard stress conditions of oxidation, photolysis and hydrolysis. The method was validated according to United States Pharmacopoeia (USP) General Chapter 〈1225〉 and was found to be selective, robust, accurate, precise, and reproducible for the simultaneous in vitro quantitation of doxycycline hyclate and its degradation impurities. The method was successfully applied for the quality assessment of current marketed doxycycline hyclate products.

In this report, a CNFs-Sm2O3 nanocomposite modified glassy carbon electrode (CNFs-Sm2O3/GCE), comprising CNFs with a large specific surface area and Sm2O3 with excellent catalytic activity, is prepared as a novel sensor for simultaneously detecting hydroquinone (HQ) and catechol (CC) in an aqueous environment. The surface characteristics and electrochemical performance of the as-prepared sensor were characterized by scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS), X-ray diffraction (XRD) and electrochemical approaches. The electrochemical results show the well-defined redox peaks of HQ and CC, and the potential difference of the peak-to-peak separation between HQ and CC is about 111 mV, as determined by both cyclic voltammetric (CV) and differential pulse voltammetric (DPV) techniques, which indicate that the modified electrodes can simultaneously determine HQ and CC without any separation step. The current of the anodic peak response of the CNFs-Sm2O3/GCE displays a linear dependence on the concentration of HQ and CC in the range of 1–500 μM, and the detection limits for HQ and CC are 0.09 and 0.07 μM, respectively. Moreover, the CNF-Sm2O3/GCE possesses excellent selectivity, reproducibility and stability in determining the concentration of the isomers of dihydroxybenzene. Therefore, this new type of electrode is promising as a sensor for simultaneous determination of HQ and CC in an aqueous environment.

BODIPY-based carbon dots as fluorescent nanoprobes for sensing and imaging of extreme acidity by Lihong Qiao; Jingwen Wang; Min Zheng; Zhigang Xie (1863-1869).
Two kinds of novel water-soluble carbon dots (CD-A and CD-B) were synthesized from 4-bora-3a,4a-diaza-s-indacene (BODIPY) derivatives, and their abilities for sensing pH under extremely acidic conditions were studied. The fluorescence of CD-A is linearly proportional to the extreme acidity range of 1.5–4.0 with a pKa of 2.90, while CD-B exhibits a linear response over the pH range of 1.0–4.0 with a pKa value of 2.63. In addition, both CD-A and CD-B were successfully applied to visualize extreme acidity in bacteria. The results demonstrate that CD-A and CD-B can serve as promising nanoprobes for the determination of extremely acidic pH values with excellent biological significance.

A sensitive fluorescence method for the detection of streptavidin based on target-induced DNA machine amplification by Mingjian Chen; Zhiyi Deng; Changbei Ma; Han Zhao; Kefeng Wu; Kemin Wang (1870-1874).
Recently, small molecule and targeted protein interactions have been applied frequently in quantitative detection, with the streptavidin (SA)–biotin model attracting most attention. We address a novel SA detection method based on the terminal protection of a small-molecule-linked DNA probe from degradation by exonuclease I and G-quadruplex-thioflavin T complex fluorescence signal amplification. With the signal amplification strategy, a limit of detection as low as 0.065 nM was reached. The developed strategy provides a new route to a low cost and highly sensitive and selective method of detection of SA in real samples.

Confirmation of the antiviral properties of medicinal plants via chemical analysis, machine learning methods and antiviral tests: a methodological approach by Tomas Drevinskas; Rūta Mickienė; Audrius Maruška; Mantas Stankevičius; Nicola Tiso; Algirdas Šalomskas; Raimundas Lelešius; Agneta Karpovaitė; Ona Ragažinskienė (1875-1885).
Medicinal plants are reported to possess antiviral activity, but finding the substances that are responsible for antiviral activity in the complex mixture of the plant extract is an extremely difficult task. In this paper a methodology related to the determination of the antiviral properties of medicinal plant extracts and based on phytochemical analysis, antiviral tests and machine learning methods is described. 16 potentially antiviral medicinal plants were selected, and their chemometric characteristics and antiviral properties were investigated. Three different analytical methods were used for chemical analysis: (i) spectrophotometry, (ii) capillary electrophoresis with contactless conductivity detection, and (iii) gas chromatography-mass spectrometry. 14 attributes were obtained describing the composition of the plant extracts. Viral growth inhibition properties were investigated and 8 candidate plant extracts were selected as being active against viruses. Infectious bronchitis virus was used as a model virus. Machine learning techniques including deep neural network classification, classification and regression tree induction and hierarchical clusterization were used for mining the factors that are responsible for antiviral effects. It was determined that (i) phenolic compounds providing high radical scavenging activity and fractions containing high content of phenolic compounds are positively related to antiviral activity in plant extracts, (ii) hydrophilic compounds that are positively charged (pKa > 4.7) in acidic media and possess medium and low electrophoretic mobility properties are negatively related to antiviral activity in medicinal plants, (iii) phenolic acids with pKa lower than 4.7 are not related to antiviral activity in the extracts, and (iv) volatile compounds in the extracts, including diversity, quantity and different volatility properties, do not affect the antiviral activity of plant extracts. Following the proposed methodological approach, it is possible to confirm which chemometric attributes are responsible for antiviral activity in medicinal plant extracts.

Voltammetric sensors based on glassy carbon electrodes (GCEs) modified with composites of polyelectrolyte complexes of chitosan (CS) and chitosan succinamide (SCS) with cyclodextrins (CDs) for the enantioselective recognition and determination of atenolol (ATN) enantiomers were studied using cyclic voltammetry and differential pulse voltammetry. The films of the polyelectrolyte complexes of CS and SCS, as well as composites with α-, β- and γ-CDs, are stable in aqueous solutions and their properties do not change over a long period of time. The analytical characteristics of the sensors, the conditions of voltammogram registration and the capabilities for the recognition and determination of ATN enantiomers were studied. It was shown that the recognition of ATN enantiomers by registering voltammograms on three GCEs modified with composites of polyelectrolyte complexes of CS and SCS with CDs, followed by chemometric treatment of the principal component analysis, resulted in a significant increase in the percentage of correctly recognised samples, in comparison with the registration of voltammograms on only one electrode. The proposed sensory system was successfully applied to the recognition and determination of ATN enantiomers in pharmaceutical formulations and human urine and to establish the manufacturers of pharmaceuticals.

A Fluorescent detection method for copper ions based on a direct redox route and desk study of wax-printed paper-based probes by Jiawei Li; Pan Li; Minghong Bian; Danqun Huo; Changjun Hou; Hui Qin; Suyi Zhang; Liang Zhang (1895-1901).
Cu2+ plays an important role in various fundamental physiological processes in organisms. Although Cu2+ probes have been developed, little research has focused on using a direct redox route to quench fluorescence for Cu2+ detection. Herein, a simple, label-free, cost-effective, and sensitive fluorescence method has been developed for Cu2+ detection. In the absence of Cu2+ and presence of NH3+ and SCN, H2O2 was employed as an oxidant to corrode BSA–Au NCs, resulting in the disappearance of fluorescence. In contrast, in the presence of Cu2+, red fluorescence was maintained because Cu(NH3)62+ can decompose H2O2. Under optimal conditions, good linear correlation between the fluorescent response and Cu2+ concentrations ranging from 5 to 1250 nM was obtained (R2 = 0.9938). Furthermore, this monitoring method has successfully been applied to real urine, tap water, and sorghum extract samples, demonstrating the potential for field applications. Based on this work, we successfully immobilized BSA–Au NCs on wax-printed paper to accomplish real-time field Cu2+ detection. Fluorescence images were captured under UV light (365 nm), and R-values (red value of RGB color model) were analyzed. The obtained linear relationship between the R-value and the Cu2+ concentration demonstrated the potential of this approach. The developed probe also showed satisfactory selectivity and excellent reproducibility. Although previous methods required a masking agent to protect against interference from increasing Hg2+ concentration, Hg2+ and other interfering ions had a near-negligible effect on results in the present work.

In this work, we have developed a rapid determination method for the low-level chemical oxygen demand (COD) index in dye-containing wastewater samples, which is based on a novel sample treatment technique that combines the advantages of microwave-assisted digestion and two-step wet-chemical oxidation with two oxidants such as KMnO4 and Ce(SO4)2. By using methylene blue (MB) as the model dye molecule, we have optimized the determination conditions and affirmed the validity of the COD assay for dye-containing wastewater. In particular, specific molar proportion relationships have been found between the added amounts of oxidants and catalysts in two kinds of digestion solutions, which exist as n(KMnO4) : n(MnSO4) = 6 : 1 and n(Ce(SO4)2) : n(Cu2+ + Ag+) = 4 : 3. Additionally, potassium tungstate as a specific reagent is introduced into the wet-chemical digestion reaction of water samples for the first time, and its positive influence on COD determination is investigated. Under optimized operating conditions, the average digestion efficiency of the MB molecule in a standard dye sample is up to 99.5%. The COD quantitative range of the MB dye is 1.0–75.0 mg O2 L−1 with a correlation coefficient (R2) of 0.9996 and a lower limit of detection (LOD) of 0.33 mg O2 per L (3σ). Moreover, the application of the method to another three types of phenothiazine dyes has also achieved satisfactory results.

Development and validation of a HPLC method to quantify DEET and IR3535 in insect repellents by Welma T. S. Vilar; Emanuella S. Sousa; Licarion Pinto; Mário César Ugulino de Araújo; Márcio José Coelho Pontes (1911-1917).
The increasing number of cases of dengue, chikungunya, yellow fever and Zika virus has caused many people to use repellents to try to protect themselves from the Aedes aegypti mosquito that transmits these diseases. There are a variety of products that have different ingredients, concentrations, durations of action and prices. However, not all repellants can be used by children and pregnant women and in some cases, medical guidance is required. Repellents are classified as cosmetics and the two most commonly used active ingredients are N,N-diethyl-3-methylbenzamide (DEET) and ethyl-3-(N-n-butyl-N-acetyl) aminopropionate (IR3535). This is a report on the development of a rapid and reliable HPLC-DAD method, validated for the quantification of DEET and IR3535 in insect repellents. The following chromatographic parameters were used: a mobile phase of water/acetonitrile at an 80 : 20 (v/v) ratio, a flow rate of 0.3 mL min−1, and a detection wavelength of 210 nm. Before analysis, samples were diluted 20 000 and 10 000 times in water/acetonitrile (90 : 10, v/v) for DEET and IR3535, respectively. The method was validated and presented satisfactory linearity (0.25–22 mg L−1, r2 = 0.9986 and 0.5–40 mg L−1, r2 = 0.9976), limit of quantification (LOQ) (0.09 and 0.06 mg L−1), with the intra- and inter-day precision calculated using a Horwitz ratio of less than 2, and recoveries (89.2–111.6% and 94.7–109.2%) for DEET and IR3535, respectively. It is important to highlight that there is no reference method that allows the quantification of both active ingredients, simultaneously, in the analyzed samples, which is proposed in this work.

Stir bar sorptive extraction and thermal desorption – gas chromatography/mass spectrometry for determining phosphorus flame retardants in air samples by Julius Matsiko; Honghua Li; Pu Wang; Huizhong Sun; Shucheng Zheng; Dou Wang; Weiwei Zhang; Yanfen Hao; Yingming Li; Qinghua Zhang; Guibin Jiang (1918-1927).
Stir bar sorptive extraction and thermal desorption coupled with gas chromatography/mass spectrometry (SBSE-TD-GC/MS) was utilized to determine phosphorus flame retardants (PFRs) in air samples. The one variable at a time (OVAT) approach was used to select the optimal conditions of the parameters that affect the SBSE procedure during thermal desorption. Desorption was carried out at 300 °C at a desorption flow rate of 76 mL min−1 for 10 minutes with the transfer line set at 320 °C. The analytes were cryofocused at −40 °C and then cryo-desorbed at 320 °C for 2 minutes. The optimal desorption conditions were then used to validate the method. The method validation showed good linearity (r2 > 0.9960), good precision (CVs ≤ 20%), low limits of detection (8.7–14.0 pg), good recoveries (>70%) and high reliability. Passive air sampling based on polydimethylsiloxane (PDMS) coated stir bars together with high volume-active air sampling (HV-AAS) (for comparison and calibration) was used to ascertain the appropriateness of the proposed method to determine the concentrations of PFRs in real air samples. The concentration results obtained by the two sampling techniques were comparable for compounds, whose passive samplers' accumulated masses were above the limits of quantitation and showed good uptake linearity over a specific period of time during the deployment. This work therefore affirms that the proposed method performs well and thus can be a suitable and cheaper alternative to active air sampling for gas phase PFRs.

A novel aptasensor for malathion blood samples detection based on DNA–silver nanocluster by Ceng Chen; Jian Shi; Yadong Guo; Lagabaiyla Zha; Lingmei Lan; Yunfeng Chang; Yanjun Ding (1928-1934).
Pesticides have drawn great attention because of their bad effects on health and the environment. Among these, organophosphates were the most widely used in China and malathion is the most common. Direct or indirect contact with malathion does harm to our nervous, respiratory and digestive systems. Thus, accurate detection of malathion in biological samples is very useful to ensure the safety of human health, especially in agricultural countries. In this paper, a sensitive and selective aptasensor based on DNA–silver nanocluster probes has been proposed for the simple and efficient detection of malathion in biological samples. First its feasibility was verified when the UV-vis absorbance apparently decreased with inclusion of malathion and the response differed from that with other chemicals. Then under optimized conditions, a concurrent decrease in absorbance with the addition of malathion from 0 μg mL−1 to 25 μg mL−1 was achieved with a limit of detection of 25 pg mL−1. The linear equations between the UV-vis absorbance spectrum and the concentration of malathion were y = 10.167x + 1.051, R2 = 0.986 (range from 0.000025 μg mL−1 to 0.025 μg mL−1) and y = 0.0136x + 1.431, R2 = 0.995 (range from 0.25 μg mL−1 to 25 μg mL−1). Moreover, investigations in blood samples were conducted to test the biocompatibility of the aptasensors for the sake of practical applications in forensic research. The detection results of the proposed method were almost consistent with those of conventional high-performance liquid chromatography (HPLC). In view of its simplicity, low cost, selectivity, sensitivity and reliability, the aptasensor introduced in our work may potentially be a better way to detect malathion in forensic toxicology examination.

V–Cr–Ti ternary alloys are important structural materials for fusion reactors, and quantification of chromium and titanium is essential in view of their significant effect on various properties of these vanadium-based alloys. Here, an X-ray fluorescence (XRF) method for analyzing (3–10)% chromium and (3–10)% titanium in a V–Cr–Ti ternary alloy has been established. The sample was prepared using two methods, namely solution and borate fusion methods. For the solution technique, the alloy sample was dissolved in a mixture of nitric acid and hydrofluoric acid, and Fe was added as an internal standard to improve precision. For the borate fusion technique, the alloy sample was dissolved in a mixture of nitric acid and hydrofluoric acid in a Pt–Au crucible, evaporated to dryness, and fused with flux to form a glass disk. Linear calibration curves were obtained by measuring synthetic standards prepared using the solution and borate fusion techniques. The results obtained were in good agreement with those given by inductively coupled plasma optical emission spectrometry (ICP-OES) and the relative standard derivation (RSD) was less than 1.0%.

Back cover (1939-1940).