Capillary Electrophoresis Analysis Research Articles

Comprehensive analysis of thalassemia alleles (CATSA) based on third-generation sequencing is a comprehensive and accurate approach for neonatal thalassemia screening

Thalassemia is one of the most common and damaging monogenic diseases in the world. It is caused by pathogenic variants of α- and/or β-globin genes, which disrupt the balance of these two protein chains and leads to α-thalassemia or β-thalassemia, respectively. Patients with α-thalassemia or β-thalassemia could exhibit a severe phenotype, with no simple and effective treatment. A three-tiered strategy of carrier screening, prenatal diagnosis and newborn screening has been established in China for the prevention and control of thalassemia, of which the first two parts have been studied thoroughly. The implementation of neonatal thalassemia screening is lagging, and the effectiveness of various screening programs has not yet been demonstrated. In this study, hemoglobin capillary electrophoresis (CE), hotspot testing method, and third-generation sequencing (TGS) were used in the variant detection of 2000 newborn samples, to assess the efficacy of these methods in neonatal thalassemia screening. Compared with CE (249, 12.45 %) and hotspot analysis (424, 21.2 %), CATSA detected the largest number of thalassemia variants (535, 26.75 %), which included 24 hotspot variants, increased copy number of α-globin gene, rare pathogenic variants, and three unreported potentially disease-causing variants. More importantly, CATSA directly determined the cis–trans relationship of variants in three newborns, which greatly shortens the clinical diagnosis time of thalassemia. CATSA showed a great advantage over other genetic tests and could become the most powerful technical support for the three-tiered prevention and control strategy of thalassemia.

Abstract 1939 High sensitivity analysis of tau aggregation models by capillary electrophoresis and Taylor dispersion analysis

Abstract 1939 High sensitivity analysis of tau aggregation models by capillary electrophoresis and Taylor dispersion analysis

α-Glucosidase immobilization on calcium-sodium alginate-chitosan gel beads for inhibitory activity evaluation from traditional Chinese medicines

Discovering α-glucosidase inhibitors is one of the effective ways to develop new anti-diabetic drugs. In this study yeast α-glucosidase immobilized in calcium alginate-chitosan gel treated with polyethyleneimine and capillary electrophoresis analysis was used to select enzyme inhibitor compounds in 12 traditional Chinese medicine. The immobilized α-glucosidase shows enhanced tolerance to temperature (70 °C), excellent repeatability (RSD = 1.9%, n = 5), high stability and good reusability (retained 57.8% and 74% of its initial activity after storing at 4 ℃ for 30 days and reusing 10 times, respectively). Moreover, the Michaelis constant of immobilized α-glucosidase and half-maximal inhibitory concentration of acarbose were calculated to be 1.79 mM and 0.70 μM, respectively. These results demonstrate that the immobilized α-glucosidase for inhibitory activity evaluation is feasible. The immobilized α-glucosidase was applied to screen inhibitors from 12 traditional Chinese medicines, and Sanguisorbae Radix the root of Sanguisorba officinalis L. exhibited the strongest inhibitory activity on α-glucosidase.

Poly(glutamic acid)-Based Viscosity Reducers for Concentrated Formulations of a Monoclonal IgG Antibody.

Above a concentration threshold, the viscosity of solutions of proteins increases abruptly, which hampers the injectability of therapeutic formulations. Concentrations above 200 g/L are an ideal goal for subcutaneous application of antibodies. Molecular additives, such as amino acids (e.g., arginine) help decrease the viscosity, but they are used at concentrations as high as about 200 mmol/L. We addressed the question of whether poly(amino acids) could be more efficient than small molecular additives. We observed marked fluidification of a model therapeutic monoclonal antibody (mAb) solution by poly(d,l-glutamic acid) and poly(l-glutamic acid) derivatives added at concentrations of <6.5 g/L (i.e., a mAb/polymer chain molar ratio between 4:1 and 1:1 mol/mol). The bare poly(glutamate) parent chains were compared with polyethylene glycol-grafted chains as PEGylation is a common way to enhance stability. Viscosity could be decreased to ∼20 mPa s as compared to values of ∼100 mPa s in the absence of polymers at 200 g/L mAb. Formation of complexes between the mAb and the polyglutamates was characterized by capillary electrophoresis analysis in dilute solutions (1 g/L mAb) and by observation of phase separation at higher concentrations, suggesting tight association at about 2:1 mol/mol mAb/polymer. Altogether, these results show that polyglutamate derivatives hold an untapped potential as an excipient for fluidification of concentrated protein solutions.

Morphological, Molecular Structural and Physicochemical Characterization of Starch Granules Formed in Endosperm of Rice with Ectopic Overexpression of α-Amylase.

The objective of this study was to characterize the endosperm starch in rice that ectopically overexpressed the α-amylase. Transgenic rice plants, transformed with cauliflower mosaic virus 35S promoter driven AmyI-1 (35S::AmyI-1) and AmyII-4 (35S::AmyII-4), and 10 kDa prolamin promoter driven AmyI-1 (P10::AmyI-1), were cultivated under standard conditions (23 °C, 12 h in the dark/ 26 °C, 12 h in the light), and brown grains were subsequently harvested. Each grain displayed characteristic chalkiness, while electron microanalyzer (EPMA)-SEM images disclosed numerous small pits on the surface of the starch granules, attributable to α-amylase activity. Fluorescence labeling and capillary electrophoresis analysis of starch chain length distribution revealed no significant alterations in the starches of 35S::AmyI-1 and 35S::AmyII-4 transgenic rice compared to the wild-type. Conversely, the extremely short α-glucan chains (DP 2-8) exhibited a dramatic increase in the P10::AmyI-1 starch. Rapid visco-analyzer analysis also identified variations in the chain length distribution of P10::AmyI-1 starch, manifesting as changes in viscosity. Moreover, 1H-NMR analysis uncovered dynamic modifications in the molecular structure of starch in rice grain transformed with P10::AmyI-1, which was found to possess unprecedented structural characteristics.

Third-generation sequencing identified two rare α-chain variants leading to hemoglobin variants in Chinese population.

Rare and novel variants of HBA1/2 and HBB genes resulting in thalassemia and hemoglobin (Hb) variants have been increasingly identified. Our goal was to identify two rare Hb variants in Chinese population using third-generation sequencing (TGS) technology. Enrolled in this study were two Chinese families from Fujian Province. Hematological screening was conducted using routine blood analysis and Hb capillary electrophoresis analysis. Routine thalassemia gene testing was carried out to detect the common mutations of α- and β-thalassemia in Chinese population. Rare or novel α- and β-globin gene variants were further investigated by TGS. The proband of family 1 was a female aged 32, with decreased levels of mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), Hb A2, and abnormal Hb bands in zone 5 and zone 12. No common thalassemia mutations were detected by routine thalassemia analysis, while a rare α-globin gene variant Hb Jilin [α139(HC1)Lys>Gln (AAA>CAA); HBA2:c.418A>C] was identified by TGS. Subsequent pedigree analysis showed that the proband's son also harbored the Hb Jilin variant with slightly low levels of MCH, Hb A2, and abnormal Hb bands. The proband of family 2 was a male at 41 years of age, exhibiting normal MCV and MCH, but a low level of Hb A2 and an abnormal Hb band in zone 12 without any common α- and β-thalassemia mutations. The subsequent TGS detection demonstrated a rare Hb Beijing [α16(A14)Lys>Asn (AAG>AAT); HBA2:c.51G>T] variant in HBA2 gene. In this study, for the first time, we present two rare Hb variants of Hb Jilin and Hb Beijing in Fujian Province, Southeast China, using TGS technology.

The establishment of a multiplex fluorescent polymerase chain reaction coupled with capillary electrophoresis analysis technology enables the simultaneous detection of 16 genotypes of human papillomavirus.

The detection and accurate genotyping of human papillomavirus (HPV) infection is critical for preventing and effectively treating cervical cancer. A multiplex fluorescent polymerase chain reaction (PCR) coupled with a capillary electrophoresis method was developed for the simultaneous detection of the 16 most prevalent HPV genotypes. Twenty-five pairs of primers were ultimately selected to ensure that both E and L regions of nine HPV genotypes, as well as the Eregions of seven HPV genotypes could be accurately amplified. This method enables the simultaneous detection and differentiation of 16 HPV genotypes in a single closed-tube reaction, accurately distinguishing products with molecular weight differences >1 bp through capillary electrophoresis. This method demonstrated exceptional accuracy, specificity, and repeatability with a detection limit of 10 copies/μL for all 16 HPV genotypes. Furthermore, 152 cervical swab specimens were obtained to compare the disparities between this approach and Cobas 4800 HPV detection method. The concordance rate and κ value were 90.1% and 0.802, respectively, indicating a high level of agreement. The established detection method was successfully applied to cervical swab specimens for determining HPV genotypes across all levels of cervical lesions, HPV52, 56, 16, and 59 were found to be most prevalent with infection rates of 10.8%, 9.1%, 6.5%, and 6.2%, respectively. This study has successfully established a detection method capable of simultaneously identifying 16 HPV genotypes. This approach can be further applied to HPV vaccine research and surveillance, with the potential for broad applications.

Metabolic Reprogramming By PRDM16 Drives Cytarabine Resistance in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) patients with high PRDM16 expression frequently experience induction failure and have a poor prognosis. However, the underlying molecular mechanisms are poorly understood. Here, we show that AML cells with high PRDM16 expression acquire resistance to cytarabine (AraC) through the activation of oxidative phosphorylation (OxPHOS) via regulation of c-MYC. Initially, we established a murine model of AML with high Prdm16 expression. We transduced c-Kit + bone marrow cells from wild-type mice with a retrovirus vector encoding MLL-AF9 (MF9) and the oncogenic short form of Prdm16 (sPrdm16). Using MF9/sPrdm16 cells, we next investigated whether AML cells with high Prdm16 expression exhibited resistance to AraC and/or daunorubicin (DNR), chemotherapeutic agents commonly used for induction chemotherapy. In the cell viability assay, MF9/sPrdm16 cells showed significantly higher IC 50 against AraC, but not against DNR, compared to MF9/control (control) cells, suggesting that high sPrdm16 expression confers relative resistance to AraC on AML cells. Colony-forming assay also confirmed resistant to AraC in MF9/sPrdm16 cells compared to control. Moreover, MF9/sPrdm16 cells were also resistant to AraC in vivo as demonstrated by the shortened survival of mice receiving MF9/sPrdm16 cells. To elucidate the mechanism of AraC resistance, we first examined cell cycle status of MF9/sPrdm16 cells since AraC exerts cytotoxicity in cell cycle dependent manner. To our surprise, MF9/sPrdm16 cells were cycling more rapidly than control cells as demonstrated by decreased G 0 phase and increased S/G 2/M phase. Intriguingly, RNA-sequencing analysis revealed that MF9/sPrdm16 cells displayed a signature of high OxPHOS by gene set enrichment analysis (GSEA). Indeed, oxygen consumption rate, superoxide production from mitochondria, and mitochondrial membrane potential were significantly elevated in MF9/sPrdm16 cells compared to control cells. The inhibition of mitochondrial respiration with metformin, the electron transport chain complex I inhibitor, or tigecycline, the mitochondrial protein synthesis inhibitor, abrogated AraC resistance in MF9/sPrdm16 cells by inducing an energetic shift towards a lower OxPHOS status. These results clearly indicated that sPrdm16 overexpression conferred MF9 cells with AraC resistance byincreasing OxPHOS in mitochondria. Next, we explored the mechanism how sPrdm16 regulates OxPHOS. In addition to elevated OxPHOS signature, RNA-sequencing data also showed strong enrichment of Myc target-signature in MF9/sPrdm16 cells. Western blot analysis showed that sPrdm16 overexpression induced up-regulation of c-Myc and its transcriptional target, Slc1a5, a glutamine transporter. We went on to analyze metabolic changes induced by sPrdm16 overexpression by capillary electrophoresis and mass spectrometry-based metabolome analysis. We found that sPrdm16 induced the accumulation of glutamine, glutamate, α-ketoglutaric acid, succinate, fumarate, and malate in MF9 cells, possibly by activating glutaminolysis via the regulation of c-Myc and Slc1a5 expression, leading to the activation of tricarboxylic acid (TCA) cycle and OxPHOS. We also confirmed the contribution of c-Myc to AraC resistance by overexpressing c-Myc in MF9 cells. As expected, MF9/c-Myc cells mimicked the phenotype of MF9/sPrdm16 cells such as resistance to AraC and high OxPHOS status. Furthermore, 10058-F4, a c-Myc inhibitor, abrogated AraC resistance in MF9/sPrdm16 cells. Next, we asked if the observation in MF9/sPrdm16 cells could be translated into human AML cells. Human AML cell lines, MOLM-13 and THP-1, with sPRDM16 overexpression also showed significantly higher IC 50 against AraC compared to Mock expressed cells. Moreover, RNA-seq data from the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG) AML-05 clinical trial showed that AML blasts with high PRDM16 expression demonstrated a gene expression signature associated with “OxPHOS” and “Myc Targets”. In conclusion, our findings demonstrate that PRDM16 overexpression activates mitochondrial respiration through metabolic reprogramming via c-MYC, thereby conferres AraC resistance on AML cells. These results suggest that targeting mitochondrial respiration might be a novel treatment strategy to overcome chemoresistance in AML patients with high PRDM16 expression.

A new device for online nanoscale sampling and capillary electrophoresis analysis of plant sap composition.

In this work, an online sampling of plant xylem sap combined with an efficient (CE)-based method was developed and applied to study the kinetics of changes in the sap composition and to assess plant fitness under stress conditions comprehensively. A laboratory-built CE device was developed to provide online sampling and CE analysis of various ionogenic species in the sap during plant stress response. The rapid online sampling and short CE analysis time allow for real-time monitoring of changes in sap constituents in the living plant during the stress response. The developed device was successfully used to analyze chloride, nitrate, and sulfate ions in the plant xylem during the salt stress or stress caused by nitrate deficiency within short time scales.

Compact contactless conductometric, ultraviolet photometric and dual-detection cells for capillary electrophoresis via additive manufacturing

The growing demand for tailored detectors in capillary electrophoresis (CE), addressing tasks like field deployment or dual-detection analysis, emphasizes the necessity for compact detection cells. In this work, we propose cost-effective and user-friendly additive manufacturing (3D-printing) approaches to produce such miniaturized detection cells suitable for a range of CE applications. Firstly, capacitively-coupled contactless conductivity detection (C4D) cells of different sizes are fabricated by casting low-melting-point alloy into 3D-printed molds. Various designs of Faraday shields are integrated within the cells and compared. A mini-C4D cell (9.5×7.0×7.5 mm3) is produced, with limits of detection for alkaline cations ranging from 8-12 μM in a short–capillary based CE application. Secondly, ultraviolet photometric (UV-PD) detection cells are fabricated using 3D printing. These cells feature two narrow slits with a width of 60 μm, which are positioned along the path of incident and transmission light to facilitate collimation. A deep UV-LED (235 nm or 255 nm) is employed as the light source, and black resin is determined to be the optimal material for 3D printing the UV-PD cell, owing to its superior UV light absorption capabilities. The UV-PD cell is connected to the LED and photodetector through two optical fibers, making it easy to switch the light source and detector. The effective pathlength and stray light percentage for detecting on a 75 μm id capillary are 74 μm and 0.5 %, respectively. Thirdly, a dual-detection cell that combined C4D and UV-PD at a single detection point is proposed. The performance of direct detection by C4D and indirect detection by UV-PD is compared for detecting organic acids. The strategies for developing cost-effective compact detection cells facilitate the versatile integration of multiple detection methods in CE analysis.

Expanded biochemical analyses of human tear fluid: Polyvalent faces of the schirmer strip

The tear film forms a protective barrier between the ocular surface and the external environment. Despite its small volume, recent advancements in preanalytical and analytical procedures have enabled its in-depth analysis using multiple approaches. However, the diversity of tear film collection methods and the lack of standardization in pre-analytical methods represent the main obstacles to reproducible results and comparison among different studies. In this study, we first improved the pre-analytical procedures for the extraction of various molecular entities from Schirmer strips (ScS). Subsequently, our investigation focused on analyzing the molecular variances that might occur between two primary tear collection methods: capillary tube (CT) and ScS. Additionally, we examined different parts of the ScS to underscore these variations, which could serve as crucial factors for developing a standardized, optimized protocol for sample processing. Our results show that the inclusion of surfactants in the extraction process enhanced both the yield of protein extraction and the number of proteins identified in ScS, by effectively lysing the cells and improving the solubility of several intracellular proteins. In addition to proteins, nucleic acids could also be recovered for gene expression analyses, particularly from the bulb region of the ScS which is placed in the cul-de-sac. Despite their diluted nature, extracts from ScS remain a suitable material for retrieving tear proteins such as IL-17A at levels as low as the fg/mL range, thanks to highly sensitive immunoassays. Collection methods can affect measured tear protein levels. Lactoferrin is found in higher percentages in capillary electrophoresis analysis of tears collected using ScS compared to tears collected by CT (39.6 ± 4.8% versus 31 ± 4.4%).

Concordance study on Y-STRs typing between SeqStudio™ genetic analyzer for HID and MiSeq™ FGx forensic genomics system

BackgroundMassively Parallel Sequencing (MPS) allowed an increased number of information to be retrieved from short tandem repeat (STR) analysis, expanding them not only to the size, as already performed in Capillary Electrophoresis (CE), but also to the sequence. MPS requires constant development and validation of the analytical parameters to ensure that the genotyping results of STRs correspond to those obtained by CE. Given the increased frequency of usage of Y-STRs as supplementary markers to the autosomal STRs analysis, it is urgent to validate the concordance of the typing results between CE and MPS analyses.Methods and resultsDNA extracted from 125 saliva samples of unrelated males was genotyped using Yfiler™ Plus PCR Amplification Kit and ForenSeq™ DNA Signature Prep Kit, which were analyzed by SeqStudio™ Genetic Analyzer for HID and MiSeq™ FGx Forensic Genomics System, respectively. For each shared Y-STR, allele designation, number of length- and sequence-based alleles per locus, stutter percentage, and the intra-locus balance of multicopy Y-STRs were screened.ConclusionsAlthough the number of forensic genetics laboratories that are applying the MPS technique in routine analysis is small and does not allow a global assessment of MPS limitations, this comparative study highlights the ability of MPS to produce reliable profiles despite the generation of large amounts of raw data.

Overview of Capillary Electrophoresis Analysis of Alkaline Phosphatase (ALP) with Emphasis on Post-Translational Modifications (PTMs)

Overview of Capillary Electrophoresis Analysis of Alkaline Phosphatase (ALP) with Emphasis on Post-Translational Modifications (PTMs)

Pediatric reference intervals of the hemoglobin fractions HbA2, HbF and HbA0 using high-performance liquid chromatography and capillary electrophoresis

Introductionβ-thalassemia is a common genetic disorder with an estimated prevalence of 80–90 million carriers worldwide. As elevated hemoglobin A2 (HbA2) is a primary feature of carriers, hemoglobin fraction analysis is a common technique used for initial screening. However, pediatric reference intervals (RIs) are scarce. Hence, the aim was to establish pediatric RIs of hemoglobin fractions using high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE). MethodsSamples were collected from assumed healthy children and adolescents of 1–18 years. Analyses were conducted using the Tosoh Automated Glycohemoglobin Analyzer HLC-723®G11 (Tosoh G11, HPLC) and the Capillarys 3 Octa (CE). Data were investigated for need of partitioning by both age (1–6 years vs. 6–18 years) and sex. ResultsIn total, 189 and 196 subjects were included in the statistical analysis of HPLC and CE, respectively. The 95% RI of HbA2 was 2.00–2.90% by HPLC and 2.2–3.0% by CE. Partitioning of data was not clinically relevant by HPLC. However, partitioning by age was suggested by CE. ConclusionRIs of hemoglobin fractions in individuals of 1–18 years using commercially available HPLC and CE equipment were reported. This is the first report of a pediatric RI of HbA2 using the Tosoh G11.

Using Cu2+ to regulate the emission feature of near-infrared fluorescent sensor with AIE: To detect ascorbic acid in food samples and its application in bioimaging

Conventional ascorbic acid (AA) detection methods such as chromatography, capillary electrophoresis, colorimetry, electrochemical detection, and enzymatic analysis require expensive equipment and complicated operation. Simple, rapid, and accurate AA detection is essential to inspect food quality, diagnose diseases, and assess immunity in humans. In this study, the first near-infrared fluorescence sensor DBHM with aggregation-induced emission was developed to detect AA under the involvement of Cu2+. The DBHM + Cu2+ sensor showed high sensitivity to AA with a limit of detection of 2.37 μM. The AA detection mechanism was investigated by optical studies, 1H NMR titration, high-resolution mass spectrometry, and infrared spectroscopy. AA was detected qualitatively and quantitatively by the DBHM + Cu2+ sensor in beverages, fruits, and Vitamin C tablets using a dual-mode (fluorescence and smartphone app) sensing platform. The new sensing system also showed low toxicity and excellent bioimaging in HeLa cells, C. elegans, and mice. This sensor could advance AA detection technology in the food industry and has potential bioimaging applications.

A rapid and reliable detection procedure of Atlantic trout introgression at the diagnostic lactate dehydrogenase chain‐1 gene

AbstractThe Italian‐native Mediterranean brown trout (Salmo ghigii) is a seriously threatened freshwater fish, especially by anthropogenic hybridisation with the domestic strains of Atlantic origin that have been repeatedly released into the wild for angling. A PCR‐restriction fragment length polymorphism (RFLP) assay of the diagnostic lactate dehydrogenase chain‐1 (LDH‐C1) gene sequences has been routinely applied to distinguish exotic from native brown trout lineages and detect Atlantic introgression signals in the Mediterranean wild populations. Here, we used dermal swab DNA obtained from 28 wild trout to improve laboratory procedures to genetically characterise trout samples at the LDH‐C1gene through (1) a capillary electrophoresis analysis of the RFLP fragments and (2) the optimisation of a diagnostic single nucleotide polymorphism analysable through mini‐sequencing approaches. The developed methods were fully consistent with those obtained through the traditional approach, but their analytical process is almost entirely automated and digitalised, thus improving result readability and accuracy in the detection of alien introgressed traces in wild Mediterranean brown trout populations.

Molecular detection of mononucleotide biomarkers of microsatellite instability in sporadic colorectal carcinoma patients with clinicopathological correlation

Objectives: To identify the frequency and types of microsatellite instability among a group of sporadic CRC patients and to correlate the findings with clinicopathological characteristics.&#x0D; Methods: During an 8-month period, all patients with sporadic CRC who attended to two teaching hospitals in Baghdad, Iraq were recruited to this cross-sectional study regardless of age, sex, ethnicity, or tumor characteristics. Demographic, clinical, and histopathological features were recorded. DNA was extracted from FFPE-blocks of the resected tumors and normal tissues. PCR amplification of five microsatellite mononucleotide repeat loci (BAT25, BAT26, NR-21, NR-24, and MONO-27) and 2 pentanucleotide repeat control markers (Penta C and Penta D) was performed to determine the MSI status. Capillary electrophoresis and Genetic Analyzer 3500 (Applied Biosystem, Japan) were used to separate and examine the products. Data were analyzed by Genescan software (Promega, USA). Instability of two or more loci is considered MSI-H.&#x0D; Result: In this study, ages of the 45 recruited patients ranged between 20-80 years, with a mean±SD of 55±12.3 years; of them, 31(68.9%) were ≥50 years; 25 (55.6%) were males. Rectal bleeding was the most frequent presenting feature [22 (48.9%)] patients; 23 (51.1%) of CRCs were located at recto-sigmoid region, 29 (64.4%) were T3 tumors, 34(75.5%) were non-mucinous adenocarcinoma, 39(86.7%) were moderately differentiated, 17 (37.8%) patients had stage III tumors; and 25 (55.5%) had lymphovascular invasion. MSI-H was seen in 5/45 (11.1%) patients; 3(60%) of them were ≥50 years, 4(80%) were males, 3(60%) were smokers, 2 (40%) presented with intestinal obstruction and altered bowel habits each; 4(80%) had T3 tumors, 3(60%) had mucinous adenocarcinomas [p=0.004], 2(40%) had stage II tumor and stage III each.&#x0D; Conclusion: The frequency of MSI-H among the recruited patients with CRC was 5/45 (11.1%) and it was significantly associated with mucinous adenocarcinoma subtype. NR-24 and NR-21 were the most prevalent instable markers.

Autonomous capillary electrophoresis processing and analysis of dried blood spots for high-throughput determination of uric acid

A new set-up for fully autonomous and high-throughput capillary electrophoresis (CE) analyses of dried blood spot (DBS) samples is presented. The DBS samples were prepared by collecting exactly 5 μL of capillary blood from a finger-prick onto a pre-punched DBS disc in a disposable plastic CE vial and by in-vial blood drying. The vials with the DBS samples were then loaded into a commercial CE instrument for a fully unmanned sample processing and analysis. A fused-silica capillary of the CE instrument was first used for the transfer of 100 μL of elution solvent to each vial, in-vial DBS elution, and in-vial eluate homogenization. The same capillary was also used for at-line injection, separation, and selective analysis of the resulting eluates. Novel CE sequences were tailor-programmed for consecutive processing and analyses of multiple DBSs, which facilitated a fully autonomous determination of uric acid with a throughput of 240 DBS samples per day (24 h). The presented analytical protocol (using 100 μm i. d./30 cm capillary; 30 mM 2-(N-morpholino)-ethanesulfonic acid, 30 mM l-histidine, and 30 μM cetyltrimethylammonium bromide background electrolyte solution; and UV detection at 292 nm) provided excellent precision at endogenous and spiked uric acid concentrations with RSD values of peak areas below 3.2%. Calibration curves were linear over the 33.3 − 1200 μM range (R2 better than 0.998), limits of detection and quantification in the original capillary blood were 10 and 33.3 μM, respectively, and were well below the uric acid clinical range (140–420 μM). The stability of uric acid in DBS samples stored at laboratory temperature for up to 2 months was also excellent demonstrating less than a 4.2% decrease in uric acid concentrations. The actual set-up might thus be highly attractive for clinical subjects and laboratories because it is minimally invasive and requires minimum intervention from laboratory staff.

Analysis of Peptides and Proteins by Native and SDS Capillary Gel Electrophoresis Coupled to Electrospray Ionization Mass Spectrometry via a Closed-Circuit Coaxial Sheath Flow Reactor Interface.

A simple and widely applicable coaxial sheath flow reactor interface (CSFRI) is introduced for easy and robust connection of liquid-phase microseparation methods to mass spectrometric detection, especially for capillary gel electrophoresis analysis of proteins and peptides including SDS-protein complexes. The interface readily accommodated post-column reactions prior to MS detection. It was demonstrated that this novel closed-circuit connection allowed the utilization of non-MS friendly buffer components without significant ion suppression and supported stable electrospray. In SDS capillary agarose gel electrophoresis mode, addition of γ-cyclodextrin to the sheath liquid efficiently removed the SDS content of the sample and the background electrolyte in the flow reactor section by inclusion complexation, while maintaining good separation efficiency and decreasing ion suppression.

Rapid quality assessment of Gentianae Macrophyllae Radix based on near infrared spectroscopy and capillary electrophoresis.

The aim of this study was to establish a rapid quality assessment method for Gentianae Macrophyllae Radix (RGM) using near infrared (NIR) spectra combined with chemometric analysis. The NIR spectra were acquired using an integrating sphere diffuse reflectance module, using air as the reference. Capillary electrophoresis (CE) analyses were performed on a model P/ACE MDQ Plus system. Partial least squares-discriminant analysis qualitative model was developed to distinguish different species of RGM samples, and the prediction accuracy for all samples was 91%. The CE response values at each retention time were predicted by building a partial least squares regression (PLSR) calibration model with the CE data set as the Y matrix and the NIR spectra data set as the X matrix. The converted CE fingerprints basically match the real ones, and the six main peaks can be accurately predicted. Transforming NIR spectra fingerprints into the form of CE fingerprints increases its interpretability and more intuitively demonstrates the components that cause diversity among samples of different species and origins. Loganic acid, gentiopicroside and roburic acid were considered as quality indicators of RGM and calibration models were built using PLSR algorithm. The developed models gave root mean square error of prediction of 0.2592% for loganic acid, 0.5341% for gentiopicroside and 0.0846% for roburic acid. The overall results demonstrate that the rapid quality assessment system can be used for quality control of RGM. This article is protected by copyright. All rights reserved.