Chromatographic Flow Research Articles

Simple and Multiplexed Detection of Nucleic Acid Targets Based on Fluorescent Ring Patterns and Deep Learning Analysis.

Simple diagnostic tests for nucleic acid targets can provide great advantages for applications such as rapid pathogen detection. Here, we developed a membrane assay for multiplexed detection of nucleic acid targets based on the visualization of two-dimensional fluorescent ring patterns. A droplet of the assay solution is applied to a cellulose nitrate membrane, and upon radial chromatographic flow and evaporation of the solvent, fluorescent patterns appear under UV irradiation. The target nucleic acid is isothermally amplified and is immediately hybridized with fluorescent oligonucleotide probes in a one-pot reaction. We established the fluorescent ring assay integrated with isothermal amplification (iFluor-RFA = isothermal fluorescent ring-based radial flow assay), and feasibility was tested using nucleic acid targets of the receptor binding domain (RBD) and RNA-dependent RNA polymerase (RdRp) genes of SARS-CoV-2. We demonstrate that the iFluor-RFA method is capable of specific and sensitive detection in the subpicomole range, as well as multiplexed detection even in complex solutions. Furthermore, we applied deep learning analysis of the fluorescence images, showing that patterns could be classified as positive or negative and that quantitative amounts of the target could be predicted. The current technique, which is a membrane pattern-based nucleic acid assay combined with deep learning analysis, provides a novel approach in diagnostic platform development that can be versatilely applied for the rapid detection of infectious pathogens.

Development of a chromatographic lateral flow immunoassay for detection of African swine fever virus antigen in blood

African swine fever (ASF) is a highly lethal disease of domestic and wild swine caused by African swine fever virus (ASFV). The disease currently circulates in Africa, Europe, Asia and on the island of Hispaniola. The ongoing epizootics in Europe and Asia have produced millions of animal deaths and severe economic losses. No effective vaccine is available for ASF, making rapid and accurate detection of ASFV essential for disease mitigation strategies. Currently available diagnostics for ASFV possess significant limitations related to assay performance, deployability, and/or turn-around time; therefore there is an unmet need for pen-side diagnostic tests with sufficient sensitivity and specificity. A chromatographic lateral flow immunoassay (LFIA) was developed for the detection of ASFV antigen in EDTA-treated whole blood using monoclonal antibodies targeting the viral p30 protein. The assay requires only water to perform and provides results in 25 min, making it well-suited for field use. The LFIA was capable of detecting genotype I and genotype II strains of ASFV in EDTA blood from experimentally infected pigs at varying time-points after infection, though it was unable to detect a genotype X ASFV strain. Diagnostic sensitivity correlated with clinical disease severity, body temperature, and viral DNA levels, and was over 90% in animals showing moderate to severe ASF-related symptoms after challenge with virulent genotype II virus. The LFIA also showed a robust diagnostic specificity of over 98%, which is essential to field testing for a high consequence to foregin animal disease. The LFIA targeting the viral p30 protein can reliably detect ASFV in whole blood from animals showing moderate to severe clinical signs of infection with virulent genotype I and II isolates, making it a promising candidate for use as a field-deployable antigen detection assay. Additional evaluation using field samples and different virus strains is required to further assess the utility of this rapid diagnostic test.

Helium-flushed sheathed nickel tube reactor for continuous flow oxygen stable isotope compound-specific analysis.

Compound-specific oxygen stable isotope analysis has been commercially available for about 20 years but the technique has been infrequently applied and practical operating knowledge is limited. Existing reactor designs and chemistry fail to fully exclude contaminating oxygen, are chromatographically flawed, and are generally fabricated from expensive platinum tubing. As a new interface between the gas chromatograph and isotope ratio mass spectrometer, we used a nickel tube reactor (containing no platinum wires) in a ceramic sheath with countercurrent helium flushing, operated at 1250°C. We analyzed suites of four long-chain alcohols (LCAs), defining the least and most 18 O-enriched LCAs as internal standards. The other two LCAs, co-injected in varying amounts, were treated as unknowns and amplitude-corrected during data reduction. With this design, signals from non-oxygenated compounds are minimized with a hydrogen trickle, while excessive graphitization is counterproductive. A balance of hydrogen and graphite is required to maintain a proper, stable redox state in the reactor. With low extraneous oxygen, the exact hydrogen flow has little effect on final δ18 O data quality. Overall average δ18 O values precisions and errors for the unknowns were better than 0.4‰ for injections between 2 and 7nmol per component. Our actively flushed sheath reduces oxygen infiltration to negligible levels and results in a chromatographic flow devoid of dead or poorly swept volumes. The improved design allows measurement of LCAs with accuracy and precision sufficient to enable investigating real processes, and may also have wider applications to other isotopes.

Influence of chromatographic column selection on the determination of 23 preservatives

以化妆品中23种防腐剂检测方法为例,探讨色谱柱选择对液相色谱方法测定结果的影响。参照《化妆品安全技术规范》甲基异噻唑啉酮等23个组分的检验方法,在2台不同的高效液相色谱仪上用15款不同品牌、型号的C18色谱柱检测23种防腐剂,计算色谱峰的理论塔板数和分离度,对23种组分的分离效果进行分析,并应用USP (United States Pharmacopeia)数据库和PQRI (Product Quality Research Institute)数据库等2种等效色谱柱选择方法,对不同色谱柱的分离效果及等效性进行评价和预测。实验结果表明,15款色谱柱对23种防腐剂的分离效果差异显著,仅有2款色谱柱可以实现23种组分的完全分离。USP和PQRI数据库中2种等效色谱柱选择方法均无法预测出合适的等效色谱柱,对23种防腐剂的液相色谱分析参考价值均较小。色谱柱是影响23种防腐剂液相色谱法测定结果准确性的关键因素,有关实验室在应用该方法时,应考虑色谱柱选择性差异。化妆品基质复杂,如何在现有研究成果的基础上,开发色谱柱的筛选和预测评价体系,进而指导实际样品的分离是下一步研究的重点、难点。建议有关部门在制修订检测方法时,注重色谱柱的耐用性考察,完善系统适应性指标,细化色谱柱分类和增加描述信息,指导色谱柱的合理选择,从而规避由于色谱柱使用过程中选择依据缺失而导致测定结果不准确的风险。

Optimizing test approaches for the detection of exposed metal surfaces within a chromatographic flow path

It is critical to the success of any chromatography-based assay that the performance of the LC instrument be checked for its readiness and ability to perform the intended analysis. This includes gaging the suitability of a system to fulfill the purpose of different types of methods. One type of analysis that requires special consideration is the analysis of compounds which are prone to a particular form of non-specific binding, namely metal adsorption, where analytes interact and potentially adsorb to metal contained within the chromatographic flow path. For an analysis of compounds which are susceptible to metal adsorption, ideally a system suitability test would be performed to ensure there will not be any sample loss or detrimental peak shape effects resulting from potential analyte-to-metal interactions. To help chromatographers assess system inertness concerns like this, we have developed a method of testing LC systems for metal interactions using adenosine 5′-(α,β-methylene)diphosphate (AMPcP). This nucleotide analog has been confirmed to have a propensity to adsorb to titanium and stainless-steel frits and is resistant to hydrolysis and stable to long-term storage and repeat use (as is befitting of any reagent proposed for system suitability testing). AMPcP has been used in a flow injection test (no column in-line) to monitor for losses in recovery and peak shape perturbations that can potentially be present in any chromatography system manufactured with one or more metal based components. In this approach, sequential injections of AMPcP were made without a column and various peak attributes were monitored and ultimately correlated to the amount of metal surface area in the flow path. The ability of this method to discriminate between inert chromatographic surfaces versus exposed metal surfaces was verified by comparing peak areas, peak shapes, and injection repeatability for AMPcP using a UHPLC equipped with MP35N metal alloy components versus an equivalent UHPLC equipped with an ethylene bridged hybrid organic-inorganic surface (or so-called hybrid surface technology). Injections of caffeine were also explored to establish a negative control for this system suitability measurement. Caffeine does not interact with metal surfaces and can therefore give an instrument specific representation of peak shape and dispersion as well as an indication of overall mechanical system performance. Additionally, replicate injections of AMPcP and caffeine onto a UHPLC partially configured with hybrid surface technology (HST) readily identified exposed metal surfaces through an increased peak area relative standard deviation as well as a reduction in absolute recovery. Finally, a novel visualization tool was developed to provide an alternative method of determining system inertness without having to perform chromatographic calculations but instead a graphical peak shape comparison between a negative control, caffeine, and the metal sensitive AMPcP test probe.

Prevalence and risk factors for helicobacter pylori infection among children in Owerri, Nigeria.

Helicobacter pylori (H. pylori) is a gram-negative ubiquitous bacterium affecting over half of the world's population. Most infections are acquired in early childhood with highest prevalence in Africa and Asia. Infected individuals develop antibodies against H. pylori which persist up to 6 months after eradication. Low socioeconomic status, poor sanitation, poor personal hygiene, and absence of potable household water supply are prevalent in our environment and also linked with increased frequency of the disease with attendant health consequences. The aim of the study was to determine the seroprevalence of H. pylori infection and potential sociodemographic factors among children in Owerri. One hundred and twenty children aged 6 months to 15 years were studied from March to June 2016. Sociodemographic data was documented in a pretested structured questionnaire while rapid test kit that detects antibody to H. pylori by chromatographic flow was used to identify subjects with infection. Data were analyzed using SPSS version 20.0 with level of statistical significance at P < 0.05. H. pylori infection prevalence was 20.0%, increased with age and highest in the 10-15 years age group (P = 0.001). Increasing age and low socioeconomic class (SEC) were found to be significant risk factors of H. pylori seropositivity among study subjects. Prevalence of H. pylori infection was high, increased with age and lower SEC as well as highest among children aged 10 years and older. Efforts should be made to exclude H. pylori infection in children with suspected symptoms considering the high burden of the disease in our setting.

Paper based analytical devices for blood grouping: a comprehensive review.

The clinical importance of blood group (BG) antigens is related to their ability to induce immune antibodies that can cause hemolysis. Yet, ABO and D (Rh) are still considered to be the key antigens for healthy blood transfusion and secondary antigens are the next priority. Serological typing is the most widely used typing method. Rapid and accurate blood grouping plays an important role in some clinical conditions, rather than conventional techniques. Hence, developing a simple and economical model for rapid blood grouping would facilitate these tests. In recent decades, paper-based microfluidics such as μPADs has gained much interest in wide application areas such as point-of-care diagnostic. In this study, we evaluated μPADs that are performed for blood grouping and its recent progress.A comprehensive literature search was performed using databases including PUBMED, SCOPUS, Web of Science and Google Scholar. Keywords were blood grouping or typing, paper analytical device, rapid test, etc.After investigation of search results, 16 papers from 2010 to 2020 were included. Further information in detail was classified in Table 1. Generally, two principles for blood typing μPADs are introduced. The lateral chromatographic flow method and the vertical flow-through method that detects BG in a visual-based manner. To detect results with acceptable clarity many factors and challenges like paper, blood sample, buffer, Ab and RBC interaction and also μPADs stability need to be considered, which are discussed. In conclusion, the simplicity, stability, cheapness, portability and biocompatibility of μPADs for blood grouping confirming its utility and also they have the capability to robust, universal blood-grouping platform. Table 1 Summary of blood grouping tests using paper-based analytical devices Antigens Type of diagnosis Validation method Sample No Accuracy Action time Paper type Stability Sample dilution Buffer Ref A, B, Rh Forward volunteers records 5 - - Whatman No. 4 - 1/2 PBS* (Khan et al. 2010) A, B, Rh Forward gel assay test and conventional slide test 100 100% 1min Whatman No. 4 and Kleeenex paper towel 7 Days in 4°C 1/1 NSS (Al-Tamimi et al. 2012) A, B, Rh Forward gel card assay 99 100% 20 Sec + Washing Kleeenex paper towel - 1/1 NSS (Li et al. 2012) A, B, Rh Forward - - - - Kleeenex paper towel - 45/100 PSS (Li et al. 2013) A, B, Rh Forward gel card assay 98 100% 1.5min Kleeenex paper towel - 85/100 PBS (Guan et al. 2014b) C, E, c, e, K, Jka, Jkb, M, N, S, P1, and Lea Forward gel card assay 266 100% - Kleeenex paper towel - 1/1 NSS (Li et al. 2014b) A, B, Rh Forward and Reverse conventional slide test 96 ≈ 91% 10min Whatman No. 1 21 Days in 4°C 1/2 NSS (Noiphung et al. 2015) C, c, E, e, K, k, Fya, Fyb, Jka, Jkb, M, N, S and s, P1, Lea and Leb Forward - 478 - - Kleeenex paper towel - 1/1 NSS, PBS (Then et al. 2015) A, B Forward and Reverse conventional slide test 76 100% 5-8min Whatman No. 4 38 Days in 4°C 1/4, 1/1 NSS (Songjaroen and Laiwattanapaisal 2016) D, K Forward volunteers records 210 - 7.5min Kleenex paper towel - 1/1 NSS (Yeow et al. 2016) A, B, c, e, D, C, E, M, N, S, s, P1, Jka, Jkb, Lea, Leb, Fya, and Fyb Forward and Reverse gel card assay 3550 ≈100% 30s Fiber glass and cotton linter 180 Days in 25°C 45/100, 1/1 PBS (Zhang et al. 2017) A, B Forward conventional slide test 598 100% 3min Whatman No. 113 14 Day in 4°C 1/1 NSS (Songjaroen et al. 2018) A, B, Rh Forward conventional slide test - - 30 Sec + Washing Unrefined sisal paper - 1/2 NSS (Casals-Terré et al. 2019) A, B, Rh Forward - - - - Whatman No.1 - 1/1 NSS (Ansari et al. 2020) ABO & Rh Forward and Reverse conventional slide test - 100% Unrefined Eucalyptus papers - 1/2 NSS, PBS (Casals-Terré et al. 2020) A, B, Rh Forward - - - 30 Sec + Washing Whatman No. 4 modified with chitosan ≥ 100days in 25°C 1/1 NSS (Parween et al. 2020) *phosphate buffer saline, normal saline solution.

Determination of two acrylates in environmental water by high performance liquid chromatography

A high performance liquid chromatography (HPLC) method was established for the determination of two acrylate substances in environmental water. The optimal chromatographic conditions were determined via exploring the effects of chromatographic column, mobile phase, column temperature, flow rate, detection wavelength and other factors on the separation effect of acrylate substances. Finally, the effective separation of methyl methacrylate and isopropyl methacrylate was realized within 6 min. The retention time of the target compound was used for qualitative analysis and the external standard method was used for quantitative analysis in the experiment. The linear relationship between the two acrylates was good in the range of 0.2-50.0 mg/L, and the correlation coefficient of standard curve was higher than 0.999. The recovery rate was 88.6%-105.3%, the relative standard deviation was 1.7%-4.1%, and the detection limit (LODs) was 0.03-0.05 mg/L. The method was simple, efficient and accurate, and suitable for the determination of acrylates in environmental water samples.

Development of a rapid screening test for Karenia mikimotoi by using loop-mediated isothermal amplification and lateral flow dipstick

As a bloom-forming dinoflagellate, Karenia mikimotoi is toxic and globally distributed. Frequent outbreaks of K. mikimotoi blooms usually lead to the mass mortality of pelagic and benthic marine life, resulting in large economic losses. Therefore, the simple, rapid, and highly effective monitoring of K. mikimotoi is crucial to minimize the damage caused by this alga. This study aimed to establish a convenient, cost-effective, and efficient molecular detection technique through loop-mediated isothermal amplification (LAMP) combined with chromatographic lateral flow dipstick (LFD) for the daily monitoring and real-time detection of K. mikimotoi. The internal transcribed spacer (ITS) and large subunit (LSU) rDNA sequences of K. mikimotoi were used to design and screen an optimal primer (KmLF3) and a detection probe (KmLF3HP). The optimized LAMP conditions were as follows: dNTP concentration, 1.2 mM; betaine concentration, 1 M; ratio of the inner primer to the outer primer, 8:1; magnesium ion concentration, 8 mM; amplification temperature, 59 °C; and amplification time, 60 min. Cross-reactivity tests confirmed the specificity of LAMP–LFD. The detection limit of LAMP–LFD for K. mikimotoi genomic DNA was 1.70 × 10−4 ng μL−1, which is 100 times lower than that of conventional PCR. The detection limit of LAMP–LFD for the recombinant plasmid containing the LSU rDNA of K. mikimotoi was 6.21 × 103 copies μL−1, which was 10 times more sensitive than that of LAMP followed by agarose gel electrophoresis and SYBR Green I dye staining and 100 times lower than that of conventional PCR. The practicability of LAMP–LFD was validated by testing with simulated field-water samples, displaying a detection limit of 10−1 cells mL−1. In conclusion, the developed LAMP–LFD is a specific, sensitive, and accurate detection method and may be competent for the field detection of K. mikimotoi.

Bioanalytical method development and validation of a liquid chromatography-tandem mass spectrometry method for determination of β-lapachone in human plasma

The purpose of this work was to develop and validate a rapid, sensitive and robust liquid chromatography tandem mass spectrometric method for the quantification of β-lapachone in human plasma and to use that method to analyze human clinical samples. Sample preparation for the developed method involved liquid-liquid extraction using ethyl acetate for extraction of β-lapachone and cryptotanshinone (internal standard) from human plasma. Chromatographic resolution was achieved on a Kinetex C18 column using a gradient elution and a chromatographic flow rate of 0.5 mL/min. The retention times of β-lapachone and cryptotanshinone were 1.98 and 2.28 min, respectively, and the method had a total run time of 4 min. Bioanalytical method validation was conducted in accordance with the United States Food and Drug Administration regulatory guidelines. The method was validated over 2 calibration ranges in order to support high- and low-dose clinical studies. Calibration curve-1 covered the range of 0.25–50 ng/mL and calibration curve-2 covered the range of 50–2000 ng/mL. The method was determined to be accurate (percent relative errors between −1.07 to 5.36 %), precise (percent relative standard deviations less than 7.4), and sensitive (LLOQ 0.25 ng/mL). β-lapachone was determined to be stable (% change from time = 0 between −11.6 and 12.6 %) across the autosampler, benchtop, freeze/thaw and long-term (63 days) stability studies. The validated bioanalytical method was employed to determine β-lapachone concentrations in human plasma samples from a clinical study.

Improvement of Loop-Mediated Isothermal Amplification Combined with Chromatographic Flow Dipstick Assay for Salmonella in Food Samples

Loop-mediated isothermal amplification (LAMP) had been employed as a powerful tool to facilitate genetic tests for various food pathogens, as it is easy to perform. Recently, various methods of detecting the LAMP amplicon were developed. In this study, we improved two LAMP assays by combining LAMP with chromatographic flow dipstick (LFD) assays for Salmonella (targeting phoP and invA, respectively). We evaluated different labeled primer sets, then selected the optimal sets to perform the LFD assays. We compared the optimal LFD and LAMP assays with the ISO standard method. The results showed that LFD was more sensitive and quicker than LAMP. Furthermore, enrichment broths of 225 food samples were tested. The sensitivity of two LFD assays was 100%. The specificity of LFD assay targeting phoP was 99.1%, and LFD assay targeting invA was 99.5%. For the LFD assay targeting phoP, the estimate of limit of detection (LOD) 50% was 0.061 CFU/g and the estimate of LOD 95% was 0.265 CFU/g. For the LFD assay targeting invA, the estimate of LOD 50% was 0.040 CFU/g and the estimate of LOD 95% was 0.172 CFU/g. We validated this method in a primary laboratory, where we accomplished the assay only using an incubator and a heating block. It suggested that the LFD assay had the potential to become a suitable diagnostic method in field test and primary labs.

Improvement and evaluation of loop-mediated isothermal amplification combined with chromatographic flow dipstick assays for Vibrio parahaemolyticus

Vibrio parahaemolyticus, a major food-borne pathogen, is a gram-negative rod-shaped halophilic bacterium which inhabits marine environments throughout the world. It can pose a threat to humans after the consumption of raw or undercooked seafood. Fast detection is crucial for hindering and controlling V. parahaemolyticus infection. Compared with traditional methods, loop-mediated isothermal amplification (LAMP) is a simple, rapid and versatile method. It can be performed at one temperature without the need for cycling. As a new method in recent years, LAMP combined with a chromatographic flow dipstick (LFD) meets the needs of point-of-care testing without the need for special instruments. It avoids the limitations of LAMP, reduces detection time and increases detection accuracy. Our previous studies have suggested that the optimized LFD method can improve the sensitivity of LAMP detection and shorten the isothermal amplification time during the detection process. In the present study, two LAMP assays were improved to LFD methods, and a LFD targeting 16S23S rRNA gene internal transcribed spacer (ITS) of V. parahaemolyticus was developed. The lower limit for tlh, toxR, ITS LFD assays were detected as 3.1 × 100, 3.1 × 101, and 3.1 × 100 CFU respectively, whether in pure cultures or artificially contaminated food samples. The shortest amplification times at the limit of each assay were determined as 20 min, 35 min and 25 min. A heating block was used to perform two (tlh and ITS) LFD assays to detect 20 food samples. Compared to a standard method (GB 4789.7–2013 National Food Safety Standards, Food Microbiology Inspection, Vibrio parahaemolyticus test), tlh and ITS LFD assays showed more MPN (most probable number) results than that of culture. It demonstrated that the improved LFD technology can provide a simple and rapid detection method with high sensitivity and specificity for detection of V. parahaemolyticus.

Improvement and evaluation of loop-mediated isothermal amplification combined with a chromatographic flow dipstick assay and utilization in detection of Vibrio cholerae

Loop-mediated isothermal amplification (LAMP) is a specific, sensitive, and easy-to-perform nucleic acid analytical technique with wide application for diagnosis of disease. Recently, LAMP combined with use of a lateral chromatographic flow dipstick (LFD) has been widely used in nucleic acid detection. However, the LFD mechanism has not been systematically analyzed, and the optimal combination of labeled primers has not been adequately evaluated. We analyzed the LAMP mechanism and discovered that the labeled loop primers played a significant role in the LFD assay. To verify our hypothesis, we developed two LFD assays for Vibrio cholerae to detect the ctxA gene and the 16S-23S ribosomal DNA internal transcribed spacer (ITS). We labeled the inner primers [forward inner primer (FIP) and backward inner primer (BIP)] and loop primers [forward loop primer (LF) and backward loop primer (LB)]. Then the labeled and unlabeled primers were combined to form ten different primer sets. We assessed the specificity, sensitivity, and efficiency of LFD assays with use of different primer compositions. All triple-labeled primer sets resulted in false positive results in the LFD assay, as did the FIP and BIP double-labeled primer set. Other double-labeled-primer sets used in LFD assays showed higher sensitivity than the LAMP assays. Moreover, FIP and LF double-labeled and BIP and LB double-labeled sets had the highest sensitivity. In both cases, assays could be performed in 20 min. We also applied the ITS LFD assays in food samples. The enrichment broths of 112 oyster samples were tested, and the proportion that tested positive by the LFD assays was 6.25%, which was not lower than the rate for the conventional PCR method (5.36%). Graphical abstract ᅟ.

Low pressure ion pair chromatography with amperometric detection for the determination of trigonelline in coffee samples

In this work, the use of ion pair chromatography strategy in low pressure chromatographic flow systems is explored for the first time. The straightforward flow manifold encompassed a peristaltic pump, an injection valve and a 1 cm-length C18 monolithic column. The amperometric detection system relied on a boron-doped diamond electrode, used as working electrode. The determination of trigonelline in coffee samples was the case-study selected. This alkaloid is an important quality marker for this commodity and is usually determined using HPLC-UV methodologies. The proposed methodology, based on ion-pair chromatography with amperometric detection, enabled the quantitative resolution of the studied analyte from the matrix compounds by adding to the mobile phase the ion pair reagent, 1-tetradecanosulfonate sodium. The present work, following the recent developments of the low pressure chromatography approach, demonstrates the potentialities of coupling monolithic columns to traditional flow analysis systems for separation and quantification of ionic or ionisable compounds.

Flow-Cell-Induced Dispersion in Flow-through Absorbance Detection Systems: True Column Effluent Peak Variance.

Following a brief overview of the emergence of absorbance detection in liquid chromatography, we focus on the dispersion caused by the absorbance measurement cell and its inlet. A simple experiment is proposed wherein chromatographic flow and conditions are held constant but a variable portion of the column effluent is directed into the detector. The temporal peak variance (σt,obs2), which increases as the flow rate (F) through the detector decreases, is found to be well-described as a quadratic function of 1/F. This allows the extrapolation of the results to zero residence time in the detector and thence the determination of the true variance of the peak prior to the detector (this includes contribution of all preceding components). This general approach should be equally applicable to detection systems other than absorbance. We also experiment where the inlet/outlet system remains the same but the path length is varied. This allows one to assess the individual contributions of the cell itself and the inlet/outlet system.to the total observed peak. The dispersion in the cell itself has often been modeled as a flow-independent parameter, dependent only on the cell volume. Except for very long path/large volume cells, this paradigm is simply incorrect.

A new UPLC approach for the quantitation of ephedrine and guaifenesin in a syrup formulation using multivariate optimization strategy

ABSTRACTA new ultraperformance liquid chromatography (UPLC) method with photodiode array detection was developed for the quantitative analysis of a commercial syrup formulation containing ephedrine (EPH) and guaifenesin (GUA). In the development of UPLC method, experimental chromatographic conditions, flow rate, column temperature, and percentage of 0.1 M H3PO4 in mobile phase, were optimized using chemometric multivariate strategy. From the application of a 33 full factorial design, the optimal chromatographic conditions were obtained as the flow rate of 0.29 mL/min, column temperature of 36.4°C, and 56.9% of 0.1 M H3PO4 in the mobile phase. The optimal conditions gave us a good chromatographic separation of the analyzed drugs with short analysis runtime within 3 min. Calibration curves for EPH and GUA in the linear working range of 4–64 and 6–96 µg/mL, respectively, were obtained using peak areas detected at 215 nm. Performance and validity of the optimized UPLC method were estimated by analyzing independent binary mixtures, inter-day and intra-day samples, and standard addition solutions containing EPH and GUA substances. It was concluded that the proposed method was a promising approach for the quantitative determination and routine analysis of a commercial syrup formulation of the titled substances.

Development of a SERS-Based Rapid Vertical Flow Assay for Point-of-Care Diagnostics.

Point-of-care (POC) diagnostic testing platforms are a growing sector of the healthcare industry as they offer the advantages of rapid provision of results, ease of use, reduced cost, and the ability to link patients to care. While many POC tests are based on chromatographic flow assay technology, this technology suffers from a lack of sensitivity along with limited capacity for multiplexing and quantitative analysis. Several recent reports have begun to investigate the feasibility of coupling chromatographic flow platforms to more advanced read-out technologies which in turn enable on-site acquisition, storage, and transmission of important healthcare metrics. One such technology being explored is surface-enhanced Raman spectroscopy or SERS. In this work, SERS is coupled for the first time to a rapid vertical flow (RVF) immunotechnology for detection of anti-HCV antibodies in an effort to extend the capabilities of this commercially available diagnostic platform. High-quality and reproducible SERS spectra were obtained using reporter-modified gold nanoparticles (AuNPs). Serial dilution studies indicate that the coupling of SERS with RVF technology shows enormous potential for next-generation POC diagnostics.

A liquid chromatography⿿atmospheric pressure photoionization tandem mass spectrometric method for the determination of organosulfur compounds in petroleum asphalt cements

We present a sensitive liquid chromatography⿿atmospheric pressure photoionization tandem mass spectrometric (UHPLC-APPI-MS/MS) method for the determination of selected organosulfur compounds in Brazilian asphalt cements. It was possible to detect 14 organosulfur compounds of different classes where sulfoxides and sulfones presented higher sensibility in ionization than thiophenes and aromatic sulfides. A dopant-assisted APPI method was also tested, however, when chromatographic flow rate was optimized a decrease in signal was observed for all compounds. PAHs were tested and ruled out as possible interfering compounds and the matrix effect of asphalt cements was within an acceptable range for the quantification of organosulfur compounds. The proposed method was found to have satisfactory linearity and accuracy with recoveries between 83.85 and 110.28% for thianaphthene and 3-methylbenzothiophene, respectively. Therefore, the method allowed the characterization of organosulfur compounds in Brazilian asphalt cements and demonstrated changes in the amount quantified in asphaltenic and maltenic fractions after the RTFOT+SUNTEST aging process.

Development of a low pressure chromatographic flow system for monitoring the biodegradation of ofloxacin and ciprofloxacin

In this work, we propose a simple low pressure chromatography method with a high throughput for monitoring the biodegradation of fluoroquinolones.

Advancement of atmospheric-vacuum interfaces for mass spectrometers with a focus on increasing gas throughput for improving sensitivity.

Ion sampling from an electrospray ionization (ESI) source was improved by increasing gas conductance of the MS inlet by 4.3-fold. Converting the gas throughput (Q) into sensitivity improvement was dependent on ion desolvation and handling of the gas load. Desolvation was addressed by using a novel slot shaped inlet that exhibited desolvation properties identical to the 0.58 mm i.d capillary. An assay tailored for "small molecules" at high chromatographic flow rate (500 μL/min) yielded a compound dependent 6.5 to 14-fold signal gain while analysis at nano chromatographic flow rate (300 nL/min) showed 2 to 3.5-fold improvement for doubly charged peptides. Improvement exceeding the Q (4.3-fold) at high chromatographic flow rate was explained by superior sampling of the spatially dispersed ion spray when using the slot shaped capillary. Sensitivity improvement across a wide range of chromatographic flow rate confirmed no compromise in ion desolvation with the increase in Q. Another improvement included less overflow of gas into the mass analyzer from the foreline region owing to the slot shape of the capillary. By doubling the roughing pump capacity and operating the electrodynamic ion funnel (EDIF) at ∼4 Torr, a single pumping stage was sufficient to handle the gas load. The transport of solvent clusters from the LC effluent into the mass analyzer was prevented by a "wavy shaped" transfer quadrupole and was compared with a benchmark approach that delivered ions orthogonally into a differentially pumped dual EDIF at comparable gas Q.