Integrated MICROFASP Method with CZE-Based Fractionation Technique and NanoRPLC-ESI-MS/MS for a Comprehensive Proteomics Analysis of a Submicrogram Sample.

We report a loss-less two-dimensional (2D) separation platform that integrated capillary zone electrophoresis (CZE) fractionation and nanoRPLC-ESI-MS/MS for a comprehensive proteomics analysis of a submicrogram sample. Protein digest was injected into the linear polyacrylamide-coated capillary, followed by CZE separation. The schemes for collecting the fractions were carefully optimized to maximize the protein coverage. The peptide fractions were directly eluted into the autosampler insert vials, followed by the nanoRPLC-ESI-MS/MS analysis without lyophilization and redissolution, thus dramatically minimizing sample loss and potential contamination. The integrated platform generated 30,845 unique peptides and 5231 protein groups from 500 ng of a HeLa protein digest within 11.5 h (90 min CZE fractionation plus 10 h LC-MS analysis). Finally, the developed platform was used to analyze the protein digest prepared by the MICROFASP method with 1 µg of cell lysate as the starting material. Three thousand seven hundred ninety-six (N = 2, RSD = 4.95%) protein groups and 20,577 (N = 2, RSD = 7.89%) peptides were identified from only 200 ng of the resulted tryptic digest within 5.5 h. The results indicated that the combination of the MICROFASP method and the developed CZE/nanoRPLC-MS/MS 2D separation platform enabled comprehensive proteome profiling of a submicrogram biological sample. Data are available via ProteomeXchange with the identifier PXD052735.

Microfluidic capillary electrophoresis - mass spectrometry for rapid charge-variant and glycoform assessment of monoclonal antibody biosimilar candidates.

Early-stage cell line screening is a vital step in developing biosimilars of therapeutic monoclonal antibodies (mAbs). While the quality of the manufactured antibodies is commonly assessed by charge-based separation methods employing UV absorbance detection, these methods lack the ability to identify resolved mAb variants. We evaluated the performance of microfluidic capillary electrophoresis coupled to mass spectrometry (MCE-MS) as a rapid tool for profiling mAb biosimilar candidates from clonal cell lines. A representative originator sample was used to develop the MCE-MS method. The addition of dimethylsulfoxide (DMSO) to the background electrolyte yielded up to 60-fold enhancement of the protein MS signal. The resulting electropherograms consistently provided resolution of mAb charge variants within 10 min. Deconvoluted mass spectra facilitated the identification of basic variants such as C-terminal lysine and proline amidation, while the acidic variants could be assigned to deamidated forms. The MCE-MS method also allowed the identification of 18 different glycoforms in biosimilar samples. To mimic early-stage cell line selection, samples from five clonal cell lines that all expressed the same biosimilar candidate mAb were compared to their originator mAb. Based on the similarity observed in charge variants and glycoform profiles acquired by MCE-MS, the most promising candidate could be selected. The MCE-MS method demonstrated good overall reproducibility, as confirmed by a transferability study involving two separate laboratories. This study highlights the efficacy of the MCE-MS method for rapid proteoform screening of clonal cell line samples, underscoring its potential significance as an analytical tool in biosimilar process development.

Analysis of cassine and spectaline in the Senna spectabilis ethanolic extracts by capillary zone electrophoresis with indirect UV detection.

INTRODUCTION: 2,6-Disubstituted piperidin-3-ols are an important group of piperidine alkaloids found in species such as Senna spectabilis, whose main constituents include cassine and spectaline, compounds with relevant pharmacological activity. The analysis of these compounds is challenging due to the complexity of plant extracts and the absence of chromophores capable of absorbing ultraviolet (UV) radiation. OBJECTIVE: This paper presents a new analytical method to separate and quantify the non-UV-absorbing alkaloids present in ethanol extracts from S. spectabilis flowers using capillary zone electrophoresis (CZE) with indirect UV detection. METHODOLOGY: The optimized CZE method employs a background electrolyte containing 60 mM histidine (His), 15 mM α-cyclodextrin, 20% acetonitrile (ACN), and pH-adjusted to 4.7 with acetic acid (AcOH). RESULTS: The limit of detection (LOD) values was 10.2 and 13.9 mg L-1 for cassine and spectaline, respectively. For both analytes, the precision data were better than 2% of relative standard deviation (RSD) for migration times and peak areas. To evaluate the applicability of the developed method, ethanolic extracts from S. spectabilis flowers were prepared and analyzed. CONCLUSIONS: Thereby, the method proved to be efficient and complementary to conventional techniques, offering a cost-effective alternative in the quantification of the non-UV-absorbing piperidine alkaloids present in plant extracts.

Quantitative analysis of therapeutic peptides by CZE using multiple sample injection in hydrodynamically closed separation system.

Therapeutic peptides have emerged as an innovative and promising class of therapeutic compounds in modern medicine. Synthetic peptide analogs triptorelin and lanreotide are known for their pronounced clinical versatility and potency. In this study, we present the development and validation of novel methods based on capillary zone electrophoresis performed in hydrodynamically closed system (HCS) and paired with ultraviolet detection and repeated injection sample introduction. To the best of our knowledge, we developed the first capillary electrophoresis-based method for the determination of lanreotide, and concurrently, the first HCS method for the determination of triptorelin. Maximal separation efficiency and signal intensity were achieved using background electrolytes composed of 50 mM formic acid with the addition of 0.05% (v/v) methyl-hydroxyethyl cellulose. The proposed methods exhibit favorable performance characteristics, namely, calibration curve (r2 exceeding 0.99), low limits of detection (0.25 µg/mL in a water matrix and 0.5 µg/mL in synthetic urine), acceptable precision (relative standard deviation ranging from 2.2% to 9.6% for intraday repeatability and between 5.2% and 14.9% for interday reproducibility), and accuracy (relative errors falling within the 91.1%-107.8% range). The method for triptorelin determination was then used for its quantification in a commercially available drug dosage form (powder for injection) and in spiked synthetic urine samples. The developed methods were also evaluated according to the novel blue applicability grade index, revealing their superior applicability. The results collectively point out the potential of the proposed methods for both quality control and clinical investigations.

Identity verification of monoclonal antibodies by triple injection capillary zone electrophoresis.

This paper presents an approach based on triple injection capillary zone electrophoresis for identification of monoclonal antibodies. The analyte to be identified is injected between two zones of a known reference. The distances between the reference zones (plug I and III) and the target zone (plug II) are adjusted by partial electrophoresis of the first and second injection plugs. The full migration time of the target analyte is calculated from the observed migration time by considering the migration times of the reference in the first and third injection plugs. The relative migration time, that is, the ratio between the full migration time of the analyte and the migration time of the reference in the third injection plug provides the basis for identification. Here, eight monoclonal antibodies, including a pair of biosimilars, were used interchangeably as both analyte and reference to investigate potential of the method. The relative migration time for a preliminary positive identification were found to vary between 0.994 and 1.006 (1.000 ± 0.006, p = 95%). Beside the relative migration time, isoform distribution, peak profiles, and early migrating peaks, originating from components in the pharmaceutical formulations, were successfully used to verify the identity of all tested monoclonal antibodies.

Development of a capillary zone electrophoresis method to monitor magnesium ion consumption during in vitro transcription for mRNA production.

Messenger RNA (mRNA) vaccines represent a landmark in vaccinology, especially with their success in COVID-19 vaccines, which have shown great promise for future vaccine development and disease prevention. As a platform technology, synthetic mRNA can be produced with high fidelity using in vitro transcription (IVT). Magnesium plays a vital role in the IVT process, facilitating the phosphodiester bond formation between adjacent nucleotides and ensuring accurate transcription to produce high-quality mRNA. The development of the IVT process has prompted key inquiries about in-process characterization of magnesium ion (Mg++) consumption, relating to the RNA polymerase (RNAP) activation, fed-batch mode production yield, and mRNA quality. Hence, it becomes crucial to monitor the free Mg++ concentration throughout the IVT process. However, no free Mg++ analysis method has been reported for complex IVT reactions. Here we report a robust capillary zone electrophoresis (CZE) method with indirect UV detection. The assay allows accurate quantitation of free Mg++ for the complex IVT reaction where it is essential to preserve IVT samples in their native-like state during analysis to avoid dissociation of bound Mg complexes. By applying this CZE method, the relationships between free Mg++ concentration, the mRNA yield, and dsRNA impurity level were investigated. Such mechanistic understanding facilitates informed decisions regarding the quantity and timing of feeding starting materials to increase the yield. Furthermore, this approach can serve as a platform method for analyzing the free Mg++ in complex sample matrices where preserving the native-like state of Mg++ binding is key for accurate quantitation.

Concentration of voxelotor in sickle cell disease can be estimated using electrophoresis and high-performance liquid chromatography.

OBJECTIVES: Voxelotor can increase hemoglobin levels in patients living with sickle cell disease (SCD). A clinician who is monitoring voxelotor response may want to know whole-blood voxelotor concentration, but this cannot be measured in most clinical settings. However, voxelotor has been demonstrated to cause "peak splitting" in common methods of hemoglobin measurement such as capillary zone electrophoresis (CZE) and high-performance liquid chromatography (HPLC). We hypothesized that we could use the size of the peak split to estimate the whole-blood concentration. METHODS: Blood from people with SCD was dosed with known concentrations of voxelotor, and multiparameter regression was used to derive the relationship of voxelotor concentration to the degree of peak splitting observed. To validate these equations, 21 patients started on voxelotor at 1500 mg/d had blood samples drawn at days 0, 14, 30, and 60. Samples were sent out for gold standard voxelotor concentration testing. The derived equations were then used to calculate voxelotor concentration. RESULTS: Calculated concentrations correlated strongly with measured concentrations for both CZE (R2 = 0.83, P < .001) and HPLC (R2 = 0.76, P < .001). Voxelotor concentration also had a significant effect on increases in hemoglobin (R2 = 0.40, P < .001). CONCLUSIONS: Thus, peak splitting CZE and HPLC can be used to estimate voxelotor concentration.

Development and Validation of a Capillary Zone Electrophoresis-Tandem Mass Spectrometry Method for Simultaneous Quantification of Eight ß-Lactam Antibiotics and Two ß-Lactamase Inhibitors in Plasma Samples.

Monitoring plasma concentrations of ß-lactam antibiotics is crucial, particularly in critically ill patients, where variations in concentrations can lead to treatment failure or adverse events. Standardized antimicrobial regimens may not be effective for all patients, especially in special groups with altered physiological parameters. Pharmacokinetic/pharmacodynamic (PK/PD) studies highlight the time-dependent antibacterial activity of these antibiotics, emphasizing the need for personalized dosing. Therapeutic drug monitoring (TDM) is essential, requiring rapid and accurate analytical methods for precise determination of drugs in biological material (typically plasma or serum). This study presents a novel capillary zone electrophoresis-tandem mass spectrometry (CZE-MS/MS) method designed for the simultaneous quantification of five penicillin antibiotics, two cephalosporins, one carbapenem, and two ß-lactamase inhibitors in a single run. The method involves a simple sample pretreatment-precipitation with organic solvent-and has a run time of 20 min. Optimization of CZE separation conditions revealed that 20 mM ammonium hydrogen carbonate (NH4HCO3) serves as the optimal background electrolyte (BGE). Positive electrospray ionization (ESI) mode, with isopropyl alcohol (IP)/10 mM ammonium formate water solution (50/50, v/v) as the sheath liquid, was identified as the optimal condition for MS detection. Method validation according to the Food and Drug Administration (FDA) guideline for development of bioanalytical methods demonstrated satisfactory selectivity, linearity, recovery, robustness, and stability. The method's practicality was evaluated using the Blue Applicability Grade Index (BAGI), yielding a score of 77.5. Moreover, the greenness of the proposed method was evaluated by two commonly used metric tools-Analytical GREEnness (AGREE) and Green Analytical Procedure Index (GAPI). The developed CZE-MS/MS method offers a practical and reliable approach for quantifying a broad spectrum of ß-lactam antibiotics in plasma. Its ability to simultaneously quantify multiple analytes in a single run, coupled with a straightforward sample pretreatment, positions it as a valuable and prospective tool for TDM in critically ill patients.

Capillary Zone Electrophoresis in Humboldt Penguins (Spheniscus humboldti).

Electrophoresis is a useful diagnostic tool for detecting inflammation, including inflammation associated with infectious diseases (eg, aspergillosis in penguins). To our knowledge, reference intervals are not available for plasma proteins via electrophoresis in Humboldt penguins (Spheniscus humboldti). Therefore, preliminary reference intervals for blood plasma proteins measured by capillary zone electrophoresis were calculated for Humboldt penguins from a single zoological collection, and possible differences between the sexes and the ages of the birds were evaluated. Lithium heparinized plasma samples from 39 Humboldt penguins were analyzed. The following sex- and age-independent reference intervals were calculated: total protein 33.8-70.4 g/L, prealbumin 1.9-4.9 g/L, albumin 12.9-31.1 g/L, albumin: globulin ratio 0.7-1.7, α-globulins 4.5-11.6 g/L, ß-globulins 5.6-20.6 g/L, and γ-globulins 2.6-8.4 g/L. Male penguins had a significantly (P = 0.047) higher albumin: globulin ratio and lower percentage of ß-globulins (P = 0.015) in comparison with female penguins. Prealbumin (g/L) significantly (P = 0.021) decreased with increased age of the penguins. These results showed some differences between the sexes and ages of the penguins, which should be considered when interpreting the results. Further studies are needed to determine whether differences in other age groups or seasons exist, and also to evaluate which infectious diseases affect plasma proteins and how the reference values calculated here may deviate in ill penguins.

A microfluidic chip-based capillary zone electrophoresis-mass spectrometry method for measuring adenosine 5'-Triphosphate and its similar nucleotide analogues.

BACKGROUND: Extracellular ATP is involved in disorders that cause inflammation of the airways and cough, thus limiting its release has therapeutic benefits. Standard luminescence-based ATP assays measure levels indirectly through enzyme degradation and do not provide a simultaneous readout for other nucleotide analogues. Conversely, mass spectrometry can provide direct ATP measurements, however, common RPLC and HILIC methods face issues because these molecules are unstable, metal-sensitive analytes which are often poorly retained. These difficulties have traditionally been overcome using passivation or ion-pairing chromatography, but these approaches can be problematic for LC systems. As a result, more effective analytical methods are needed. RESULTS: Here, we introduce a new application that uses microfluidic chip-based capillary zone electrophoresis-mass spectrometry (µCZE-MS) to measure ATP and its analogues simultaneously in biofluids. The commercially available ZipChip Interface and a High-Resolution Bare-glass microchip (ZipChip, HRB, 908 Devices Inc.) coupled to a Thermo Scientific Tribrid Orbitrap, were successfully used to separate and detect various nucleotide standards, as well as ATP, ADP, AMP, and adenosine in plasma and BALF obtained from naïve Brown Norway rats. The findings demonstrate that this approach can rapidly and directly detect ATP and its related nucleotide analogues, while also highlighting the need to preserve these molecules in biofluids with chelators like EDTA. In addition, we demonstrate that this µCZE-MS method is also suitable for detecting a variety of metabolites, revealing additional potential future applications. SIGNIFICANCE: This innovative µCZE-MS approach provides a robust new tool to directly measure ATP and other nucleotide analogues in biofluids. This can enable the study of eATP in human disease and potentially contribute to the creation of ATP-targeting therapies for airway illnesses.

Pilot Evaluation of the Long-Term Reproducibility of Capillary Zone Electrophoresis-Tandem Mass Spectrometry for Top-Down Proteomics of a Complex Proteome Sample.

Mass spectrometry (MS)-based top-down proteomics (TDP) has revolutionized biological research by measuring intact proteoforms in cells, tissues, and biofluids. Capillary zone electrophoresis-tandem MS (CZE-MS/MS) is a valuable technique for TDP, offering a high peak capacity and sensitivity for proteoform separation and detection. However, the long-term reproducibility of CZE-MS/MS in TDP remains unstudied, which is a crucial aspect for large-scale studies. This work investigated the long-term qualitative and quantitative reproducibility of CZE-MS/MS for TDP for the first time, focusing on a yeast cell lysate. Over 1000 proteoforms were identified per run across 62 runs using one linear polyacrylamide (LPA)-coated separation capillary, highlighting the robustness of the CZE-MS/MS technique. However, substantial decreases in proteoform intensity and identification were observed after some initial runs due to proteoform adsorption onto the capillary inner wall. To address this issue, we developed an efficient capillary cleanup procedure using diluted ammonium hydroxide, achieving high qualitative and quantitative reproducibility for the yeast sample across at least 23 runs. The data underscore the capability of CZE-MS/MS for large-scale quantitative TDP of complex samples, signaling its readiness for deployment in broad biological applications. The MS RAW files were deposited in ProteomeXchange Consortium with the data set identifier of PXD046651.

Proteomics Parameters for Assessing Authenticity of Grated Grana Padano PDO Cheese: Results from a Three-Year Survey.

Assessing the authenticity of PDO cheeses is an important task because it allows consumer expectations to be fulfilled and guarantees fair competition for manufacturers. A 3-year survey was carried out, analyzing 271 samples of grated Grana Padano (GP) PDO cheese collected on the European market. Previously developed analytical methods based on proteomics approaches were adopted to evaluate the compliance of market samples with selected legal requirements provided by the specification for this cheese. Proteolysis follows highly repeatable pathways in GP cheese due to the usage of raw milk, natural whey starter, and consistent manufacturing and ripening conditions. From selected casein breakdown products, it is possible to calculate the actual cheese age (should be >9 months) and detect the presence of excess rind (should be <18%). Furthermore, due to the characteristic pattern of free amino acids established for GP, distinguishing it from closely related cheese varieties is feasible. Cheese age ranged from 9 to 25 months and was correctly claimed on the label. Based on the amino acid pattern, three samples probably contained defective cheese and there was only one imitation cheese. Few samples (9%) were proven to contain some excess rind. Overall, this survey highlighted that the adopted control parameters can assure the quality of grated GP.

CE methods for charge variant analysis of mAbs and complex format biotherapeutics.

Charge heterogeneity analysis of monoclonal antibodies (mAbs) and complex formats, such as bispecifics, is crucial for therapeutic applications. In this study, we developed two capillary electrophoresis (CE)-based methods, capillary zone electrophoresis (CZE) and imaged capillary isoelectric focusing (iCIEF), for analyzing a broad spectrum of mAbs and complex mAb formats. For CZE, we introduced a new buffer system and optimized the background electrolyte (BGE) with an alternative dynamic coating agent and a superior polymeric additive. The pH of the BGE was increased, leading to enhanced resolution of high pI and complex format mAbs. In iCIEF, we identified an ampholyte combination offering a highly linear pH gradient and covering a suitable pH range. We also investigated alternatives to denaturing stabilizers and found that non-detergent sulfobetaine 195 exhibited excellent properties for iCIEF applications. These optimized methods provide a framework for the charge heterogeneity analysis of therapeutic mAbs and complex formats.

A comparative evaluation of the analytical performances of premier resolution-high-performance liquid chromatography (PR-HPLC) with capillary zone electrophoresis (CZE) assays for the detection of hemoglobin variants and the quantitation of HbA0, A2, E, and F.

OBJECTIVES: Hemoglobinopathies, including thalassemia and hemoglobin (Hb) variants, are common hematological disorders in tropical countries. Accurate and precise separation of hemoglobin types and reliable quantitation are necessary for differential diagnosis of these disorders. METHODS: We have evaluated the analytical performances of premier resolution-high-performance liquid chromatography (PR-HPLC; Trinity Biotech, Co. Wicklow, Ireland) to assist in the presumptive diagnosis of thalassemia and Hb variants commonly found in Southeast Asian countries. HbA0, HbA2, HbE, and HbF levels were separated and quantified in 120 blood samples from unrelated adult subjects and compared with those analyzed by capillary zone electrophoresis (CZE; CAPILLARYS™ 2, Sebia, Norcross, GA, US). The Hb analysis patterns of Hb variants obtained from the PR-HPLC system were also compared to those obtained from HPLC (VARIANT II, ß-thalassemia Short Program, Bio-Rad, Laboratories, Hercules, CA, US) and CZE systems. RESULTS: The PR-HPLC had excellent precision with a coefficient of variation (CV) for HbA2 quantitation of 3.8 % within-run and 5.2 % between-run. The levels of HbA2/E quantified by the PR-HPLC system correlated well with those of the CZE system (r=0.997). In addition, thalassemia interpretation results obtained from the PR-HPLC and the CZE showed 100 % agreement. Moreover, chromatograms of the PR-HPLC were also comparable to those of VII-HPLC and CAP2-CZE electropherograms. CONCLUSIONS: The PR-HPLC system would be applicable to diagnose common forms of thalassemia and Hb variants in Southeast Asia.

CZE-MS peptide mapping: To desalt or not to desalt?

BACKGROUND: In "shotgun" approaches involving high-performance liquid chromatography or capillary zone electrophoresis (CZE), matrix removal prior to sample analysis is considered as an indispensable tool. Despite the fact that CZE offers a high tolerance towards salts, most publications reported on the use of desalting. There seems to be no clear consensus on the utilization of desalting in the CZE-MS community, most probably due to the absence of works addressing the comparison of desalted and non-desalted digests. Our aim was to fill this research gap using protein samples of varying complexity in different sample matrices. RESULTS: First, standard protein digests were analyzed to build the knowledge on the effect of sample clean-up by solid-phase extraction (SPE) pipette tips and the possible stacking phenomena induced by different sample matrices. Desalting led to a somewhat altered peptide profile, the procedure affected mostly the hydrophilic peptides (although not to a devastating extent). Nevertheless, desalting samples allowed remarkable stacking efficiency owing to their low-conductivity sample background, enabling a so-called field-amplified sample stacking phenomenon. Non-desalted samples also produced a stacking event, the mechanism of which is based on transient-isotachophoresis due to the presence of high-mobility ions in the digestion buffer itself. Adding either extra ammonium ions or acetonitrile into the non-desalted digests enhanced the stacking efficiency. A complex sample (yeast cell lysate) was also analyzed with the optimal conditions, which yielded similar tendencies. SIGNIFICANCE: Based on these results, we propose that sample clean-up in the bottom-up sample preparation process prior to CZE-MS analysis can be omitted. The preclusion of desalting can even enhance detection sensitivity, separation efficiency or sequence coverage.

A methodological approach by capillary electrophoresis coupled to mass spectrometry via electrospray interface for the characterization of short synthetic peptides towards the conception of self-assembled nanotheranostic agents.

Nanostructures formed by the self-assembling peptide building blocks are attractive materials for the design of theranostic objects due to their intrinsic biocompatibility, accessible surface chemistry as well as cavitary morphology. Short peptide synthesis and modification are straightforward and give access to a great diversity of sequences, making them very versatile building blocks allowing for the design of thoroughly controlled self-assembled nanostructures. In this work, we developed a new CE-DAD-ESI-MS method to characterize short synthetic amphiphilic peptides in terms of exact sequence and purity level in the low 0.1 mg.mL-1 range, without sample treatment. This study was conducted using a model sequence, described to have pH sensitive self-assembling property. Peptide samples obtained from different synthesis processes (batch or flow, purified or not) were thus separated by capillary zone electrophoresis (CZE). The associated dual UV and MS detection mode allowed to evidence the exact sequence together with the presence of impurities, identified as truncated or non-deprotected sequences, and to quantify their relative proportion in the peptide mixture. Our results demonstrate that the developed CE-DAD-ESI-MS method could be directly applied to the characterization of crude synthetic peptide products, in parallel with the optimization of peptide synthetic pathway to obtain controlled sequences with high synthetic yield and purity, which is crucial for further design of robust peptide based self-assembled nanoarchitectures.

Analysis of full and empty ratio of EV71 virus by using capillary zone electrophoresis.

Hand, foot, and mouth disease is a serious public health problem, and the main pathogen is enterovirus 71 (EV71). Its capsid assembly mechanism including capsid protein processing has been widely studied. Full and empty capsids have different immunological efficacy. Therefore, tracking full/empty capsid ratio throughout the EV71 production process is important to ensure consistent product quality and proper dosing response. The analysis of full/empty capsid ratio of intact virus has been widely reported as well. A variety of techniques have been employed to evaluate the full/empty capsid ratios. However, there has not been a rapid, reproducible, and robust assay to determine the full/empty capsid ratios of final and in-process products. In this study, a novel assay based on capillary zone electrophoresis was established. The separation of full and empty species could be achieved within 10 min and the ratio of peak areas was used to calculate the full/empty capsid ratio directly. The results showed good reproducibility and linearity for the determination of full/empty capsid ratios.

Monoclonal Light Chains with alpha 2 mobility on Serum Protein Electrophoresis.

Multiple myeloma (MM) is a neoplasm characterized by malignant proliferation of plasma cells that produce excessive quantities of a single type of immunoglobulin (Ig) called as monoclonal immunoglobulin or M-protein or paraprotein. M-protein produced can be either an intact antibody with both heavy and light-chain components or only light chains or rarely only heavy chains. Presence of M-protein in serum protein electrophoresis (PEP) is useful in the diagnosis, prognosis, and treatment of MM and other plasma cell dyscrasias. These M-proteins are identified commonly in beta and gamma regions and very rarely in alpha 2 region, appearing as a narrow band in agarose electrophoresis or as a sharp symmetric spike (M-spike) or peak in capillary zone electrophoresis. Here, we present an unusual case of monoclonal light chains producing two M- spikes in the alpha 2 globulin region in capillary zone electrophoresis.

Quantitation of native and forced degraded collagens by capillary zone electrophoresis: Method development and validation.

A new capillary zone electrophoresis method for collagen quantitation was developed and validated according to the International Council for Harmonization guideline Q2 (R1). The Sircol collagen assay and ultraviolet spectrometry were employed as reference methods. Capillary zone electrophoresis enables specific, simple, and fast determination within 9 min. It is less user-dependent and more automated than the Sircol collagen assay. With a limit of detection of 18.0 µg/mL, the new method is less sensitive than the Sircol collagen assay, which has a limit of detection of 6.5 µg/mL. Nonetheless, capillary zone electrophoresis covers a wider linearity range (50-400 µg/mL) compared to the Sircol collagen assay (5-80 µg/mL), with similar precision. Additional advantages of capillary zone electrophoresis are the ability to gain information on collagen integrity and to simultaneously determine native and denatured collagens. This approach represents a modern and legitimate alternative to the Sircol collagen assay. The developed method has been successfully applied to the study of three collagen products and samples from forced degradation.

Comparison of HbA2 Using High Performance Liquid Chromatography Versus Haemoglobin Capillary Zone Electrophoresis.

Thalassemia is among the most common hereditary disorders in the world. Approximately 5% of the world's population are carriers of hemoglobinopathies, and 2.9% are carriers of beta thalassemia. Haemoglobin A2 (HbA2) constitutes less than 3% of the total hemoglobin (Hb) in adults, and the determination of Hb A2 levels is important to diagnose the beta thalassemia trait (BTT). In some cases, the level of HbA2 is not typically elevated, and some difficulties may arise in making the diagnosis. Cation exchange high-performance liquid chromatography (HPLC) and HbCZE (haemoglobin capillary zone electrophoresis) are considered acceptable methods to diagnose BTT, but these vary in their accuracy and cut-offs. In this study, we attempted to compare HbA2 values using two methods, HPLC and HbCZE, in 536 whole blood samples sent by physician-ordered hemoglobinopathy screening over two years. This included antenatal women, patients with anemia not responding to iron, and cases of familial screening where either a child or a sibling had been diagnosed with hemoglobinopathy or thalassemia. The performance characteristics of both machines were compared for the detection of the 5 most common hemoglobin variants: Hb A, HbF, HbS, Hb C, and HbE. On comparing the HbA2 values, the HPLC showed higher values for HbA2 as compared to HbCZE, while the HbF and HbS measurement agreement was good between both methods. Normal ranges and mean normal values of HbA2 differ between different methods and different manufacturers; hence, each institute using these machines should validate its cutoffs.