Application of an optimized interpretation model in capillary hemoglobin electrophoresis for newborn thalassemia screening.

INTRODUCTION: Newborn screening is an important supplement to thalassemia control and prevention. Capillary electrophoresis (CE) technology has several advantages for thalassemia screening but with low sensitivity, especially for thalassemia carriers. This study aims to illustrate the application of an optimized interpretation model in newborn thalassemia screening by capillary hemoglobin electrophoresis. METHODS: Two thousand, two hundred fifty-eight neonates selected from four regions in China were enrolled and were screened for α-thalassemia and ß-thalassemia by capillary electrophoresis. Results were interpreted based on an optimized model integrated with multiple parameters. Molecular analysis was carried out in synchrony and used as the gold standard for the screening performance assessment. The consistency among different regions and thalassemia genotypes were also investigated. RESULTS: Among the 2258 neonates, 485 were identified to have a likely diagnosis of thalassemia, and 422 α-thalassemia, 80 ß-thalassemia, and 21 α/ß-thalassemia cases were confirmed by molecular analysis, including 277 α-thalassemia silent carriers, 135 α-thalassemia trait carriers, 10 Hemoglobin H disease, and 80 ß-thalassemia trait carriers. The screening sensitivity, specificity, positive, and negative predictive value for α-thalassemia and ß-thalassemia were 84.83%, 99.14%, 95.98%, 96.41%, and 88.75%, 98.73%, 76.34%, and 99.48%, respectively. The optimized interpretation model showed higher performance for thalassemia carriers, though some neonates with silent α-thalassemia genotypes (-α3.7 /αα, -α4.2 /αα, and αWS α/αα) and ß-28 /ßN genotype were still missed. The screening performance among different regions was comparable. CONCLUSIONS: Capillary hemoglobin electrophoresis with the optimized interpretation model shows reliable performance for newborn thalassemia screening. It is applicable to large-scale population screening.

Endpoint PCR coupled with capillary electrophoresis (celPCR) provides sensitive and quantitative measures of environmental DNA in singleplex and multiplex reactions.

The use of sensitive methods is key for the detection of target taxa from trace amounts of environmental DNA (eDNA) in a sample. In this context, digital PCR (dPCR) enables direct quantification and is commonly perceived as more sensitive than endpoint PCR. However, endpoint PCR coupled with capillary electrophoresis (celPCR) potentially embodies a viable alternative as it quantitatively measures signal strength after PCR in Relative Fluorescence Units (RFU). Provided comparable levels of sensitivity are reached, celPCR permits the development of cost-efficient multiplex reactions, enabling the simultaneous detection of several target taxa. Here, we compared the sensitivity of singleplex and multiplex celPCR to dPCR for species-specific primer pairs amplifying mitochondrial DNA (COI) of fish species occurring in European freshwaters by analyzing dilution series of tissue extracts as well as field-collected water samples. Both singleplex and multiplex celPCR and dPCR displayed comparable sensitivity with reliable positive amplifications starting at two to 10 target DNA copies per µl extract. celPCR was suitable for quantifying target DNA and direct inference of copy numbers from RFU was possible after accounting for primer effects in linear mixed-effects models and calibration via dPCR. Furthermore, multiplex celPCR and dPCR were successfully used for the detection and quantification of fish-eDNA in field-collected water samples, confirming the results of the dilution series experiment and exemplifying the high sensitivity of the two approaches. The possibility of detection and quantification via multiplex celPCR is appealing for the cost-efficient screening of high sample numbers. The present results confirm the sensitivity of this approach thus enabling its application for future eDNA-based monitoring efforts.

Reference intervals for electrophoretograms obtained by capillary electrophoresis of dialyzed urine from healthy dogs.

Electrophoresis of urine to evaluate protein fractions in dogs with proteinuria to differentiate glomerular from tubular damage has increased in recent years; however, capillary electrophoresis (CE) of urine has not been reported in a study of > 40 healthy animals, to our knowledge. We aimed to establish reference intervals (RIs) for the urine protein fractions obtained by CE of urine from healthy dogs. We obtained urine samples from 123 clinically healthy dogs of both sexes between December 2016 and April 2019; urine was frozen until CE was performed. The electrophoretic patterns obtained were divided into 5 protein fractions, and RIs were established in percentages and absolute values using nonparametric methods. RIs were obtained for the fractions (F) as follows: 5.5 to 56.2% for F1, 3.2 to 16.5% for F2, 3.5 to 16.2% for F3, 17.8 to 69.8% for F4, and 5.1 to 23.9% for F5. These RIs obtained by CE might be useful clinically as a basis for comparison with pathologic samples. Age was a statistically significant factor for F2 (p = 0.01) and F3 (p = 0.02), and sex was a statistically significant factor for F1 (p = 0.03).

The profiling of microbiota in vaginal swab samples using 16S rRNA gene sequencing and IS-pro analysis.

BACKGROUND: 16S rRNA gene sequencing is currently the most common way of determining the composition of microbiota. This technique has enabled many new discoveries to be made regarding the relevance of microbiota to the health and disease of the host. However, compared to other diagnostic techniques, 16S rRNA gene sequencing is fairly costly and labor intensive, leaving room for other techniques to improve on these aspects. RESULTS: The current study aimed to compare the output of 16S rRNA gene sequencing to the output of the quick IS-pro analysis, using vaginal swab samples from 297 women of reproductive age. 16S rRNA gene sequencing and IS-pro analyses yielded very similar vaginal microbiome profiles, with a median Pearson's R2 of 0.97, indicating a high level of similarity between both techniques. CONCLUSIONS: We conclude that the results of 16S rRNA gene sequencing and IS-pro are highly comparable and that both can be used to accurately determine the vaginal microbiota composition, with the IS-pro analysis having the benefit of rapidity.

The maternal serum metabolome by multisegment injection-capillary electrophoresis-mass spectrometry: a high-throughput platform and standardized data workflow for large-scale epidemiological studies.

A standardized data workflow is described for large-scale serum metabolomic studies using multisegment injection-capillary electrophoresis-mass spectrometry. Multiplexed separations increase throughput (<4 min/sample) for quantitative determination of 66 polar/ionic metabolites in serum filtrates consistently detected (coefficient of variance (CV) <30%) with high frequency (>75%) from a multi-ethnic cohort of pregnant women (n = 1,004). We outline a validated protocol implemented in four batches over a 7-month period that includes details on preventive maintenance, sample workup, data preprocessing and metabolite authentication. We achieve stringent quality control (QC) and robust batch correction of long-term signal drift with good mutual agreement for a wide range of metabolites, including serum glucose as compared to a clinical chemistry analyzer (mean bias = 11%, n = 668). Control charts for a recovery standard (mean CV = 12%, n = 2,412) and serum metabolites in QC samples (median CV = 13%, n = 202) demonstrate acceptable intermediate precision with a median intraclass coefficient of 0.87. We also report reference intervals for 53 serum metabolites from a diverse population of women in their second trimester of pregnancy.

Stability study of α-bromophenylacetic acid: Does it represent an appropriate model analyte for chiral separations?

The stability of α-bromophenylacetic acid (BPAA) in 50% aqueous methanol solution has been tested. CE in different running buffers was used to separate BPAA from the decomposition reaction products α-hydroxyphenylacetic (mandelic) acid and α-methoxyphenylacetic acid. Suitable CE separation of all three compounds and other product, bromide, was achieved in 60 mmol/L formate buffer (pH 3.0) at -30 kV in 50 µm (i.d.) poly(vinyl alcohol)-coated fused silica capillary (30 cm/24.5 cm) with UV detection at 200 nm. The CE method was applied to determine the reaction order of the decomposition of BPAA (0.47 mmol/L) via nucleophilic substitution in 50% aqueous methanol. The first-order reaction kinetics was confirmed by linear and non-linear regression, giving the rate constants 1.52 × 10-4 ± 2.76 × 10-5 s-1 and 7.89 × 10-5 ± 5.02 × 10-6 s-1, respectively. Additionally, the degradation products were identified by CE coupled to mass spectrometric (MS) detection. The CE-MS experiments carried out in 60 mmol/L formate buffer (pH 3.0) and in 60 mmol/L acetate buffer (pH 5.0) confirmed the results obtained by CE-UV. Furthermore, the stability of BPAA in polar solvents was tested by 1H NMR experiments. Our results provide strong evidence of the instability and fast degradation of BPAA in 50% aqueous methanol indicating that BPAA is not suitable as the model analyte for chiral separations.

Impact of Pre-Analytical Factors on MSI Test Accuracy in Mucinous Colorectal Adenocarcinoma: A Multi-Assay Concordance Study.

Immunohistochemistry (IHC) and polymerase chain reaction (PCR) and fragment separation by capillary electrophoresis represent the current clinical laboratory standard for the evaluation of microsatellite instability (MSI) status. The importance of reporting MSI status in colorectal cancer is based on its potential for guiding treatment and as a prognostic indicator. It is also used to identify patients for Lynch syndrome testing. Our aim was to evaluate pre-analytical factors, such as age of formalin-fixed and paraffin-embedded (FFPE) block, neoplastic cell percentage, mucinous component, and DNA integrity, that may influence the accuracy of MSI testing and assess the concordance between three different MSI evaluation approaches. We selected the mucinous colorectal cancer (CRC) histotype for this study as it may possibly represent an intrinsic diagnostic issue due to its low tumor cellularity. Seventy-five cases of mucinous CRC and corresponding normal colon tissue samples were retrospectively selected. MMR proteins were evaluated by IHC. After DNA quality and quantity evaluation, the Idylla™ and TapeStation 4200 platforms were adopted for the evaluation of MSI status. Seventy-three (97.3%) cases were successfully analyzed by the three methodologies. Overall, the Idylla™ platform showed a concordance rate with IHC of 98.0% for microsatellite stable (MSS)/proficient MMR (pMMR) cases and 81.8% for MSI/deficient MMR (dMMR) cases. The TapeStation 4200 system showed a concordance rate with IHC of 96.0% for MSS/pMMR cases and 45.4% for MSI/dMMR cases. The concordance rates of the TapeStation 4200 system with respect to the Idylla™ platform were 98.1% for MSS profile and 57.8% for MSI profile. Discordant cases were analyzed using the Titano MSI kit. Considering pre-analytical factors, no significant variation in concordance rate among IHC analyses and molecular systems was observed by considering the presence of an acellular mucus cut-off >50% of the tumor area, FFPE year preparation, and DNA concentration. Conversely, the Idylla™ platform showed a significant variation in concordance rate with the IHC approach by considering a neoplastic cell percentage >50% (p-value = 0.002), and the TapeStation 4200 system showed a significant variation in concordance rate with the IHC approach by considering a DNA integrity number (DIN) ≥4 as cut-off (p-value = 0.009). Our data pinpoint a central role of the pre-analytical phase in the diagnostic outcome of MSI testing in CRC.

Application of multisegment injection on quantification of creatinine and standard addition analysis of urinary 5-hydroxyindoleacetic acid simultaneously with creatinine normalization.

In this paper, the development of a simple dilute-and-shoot method for quantifying urinary creatinine by CE-ESI-MS was described. The creatinine analysis time was about 7 min/sample by conventional single injection (SI) method and can be significantly reduced to less than 2 min/sample with multi-segment injection (MSI). In addition, the standard addition analysis of 5-hydroxyindole-3-acetic acid (5-HIAA) and creatinine normalization was performed within one run by the MSI technique, and the total analysis time was 14-min faster compared to the SI method for analyzing the same set of samples. The uses of isotopic and non-isotopic internal standards (ISs) were compared. Creatinine-(methyl-13 C) and 5-hydroxyindole-4,6,7-D3 -3-acetic-D2 acid (5-HIAA-D5 ) used as isotopic ISs can provide both accurate and precise results. In contrast, 1,5,5-trimethylhydantoin (1,5,5-TH) used as the non-isotopic IS for creatinine may cause a bias of over 13% in SI method and even worse when the MSI technique was used. Another compound, 2-methyl-3-indoleacetic acid (2-MIAA), was determined not suitable for MSI analysis of 5-HIAA due to endogenous interferences despite its acceptable performance in conventional methods of analysis.

A Pitfall in HbA1c Testing Caused by Hb Long Island Hemoglobin Variant.

BACKGROUND: Although many factors may interfere with hemoglobin (Hb)A1c measurement, Hb variants are among the most important factors. METHODS: We tested the HbA1c levels of the patient, a 32 year old Manchu Chinese woman, during a routine health check. We used different methods, including high-performance liquid chromatography (HPLC) and capillary electrophoresis, to test specimens from the patient. Next, we tested the specimen further using polymerase chain reaction (PCR) and sequencing. RESULTS: We discovered that our patient, who had an HbA1c value of 0, also has an Hb variant, Hb Long Island, which we found during the HbA1c analysis as part of her routine health check at the Health Management Center in the General Hospital of Tianjin Medical University, Tianjin, China. Also, we discovered that the exon 1 of ß gene contained transversion mutations, with 1 heterozygous and 1 homozygous variant (HBB:c.8A > C, 9T > C). These gene mutations resulted in an amino-acid change (His to Pro) and a decrease in HbA1c value. CONCLUSIONS: When there is no correlation between the clinical signs, glycemic status, and glycated Hb levels of the patient, the chromatogram of HbA1c should be carefully checked to detect possible variants that cause interference in the measurement.

Capillary hemoglobin electrophoresis of healthy and anemic dogs: Quantification, validation, and reference intervals of hemoglobin fractions.

Despite the advances in canine medicine and the rapid gaining of attention of canine models in biomedical field and particularly in hemoglobin genes research, the studies on canine hemoglobin composition are sparse with ambiguous findings. Our aim was: i) to investigate the electrophoretic pattern of canine hemoglobin and the possible effects of age, sex, and anemia using a capillary electrophoresis assay, and ii) to validate this assay and calculate reference intervals (RIs) for canine hemoglobin fractions. Blood samples were collected from 53 healthy and 42 dogs with regenerative and non-regenerative anemias. The Sebia Capillarys 2 flex-piercing was used for hemoglobin analysis and it was validated using canine blood samples. R statistical language was employed for the statistical analyses. A major hemoglobin fraction (named HbA0) and a minor one (named HbA2) were identified in 100% and 47.4% of samples, respectively. The within-run and between-run CV was 0.1% for HbA0, and 9.1% and 11.2% for HbA2, respectively. The extremely narrow range of HbA0 and HbA2 values hampered a linearity study using canine blood samples. The RIs for HbA0 and HbA2 were 98.9-100% and 0-1.1%, respectively. HbA0 and HbA2 values were not significantly correlated with age (P = 0.866) or reticulocyte count (P = 0.731). No differences were observed in the median HbA0 and HbA2 between the two sexes (P = 0.887), and healthy and anemic dogs (P = 0.805). In conclusion, the capillary electrophoresis revealed a major hemoglobin fraction and an inconsistently present minor fraction. No effect of age, sex, anemia, or regenerative status of anemia was detected. The assay used was validated and RIs were generated, so as to be suitable for use in future investigations.

Carbohydrate-deficient transferrin: utility of HPLC in handling atypical samples uninterpretable by capillary electrophoresis.

AIMS: Carbohydrate-deficient transferrin (CDT) is a marker of chronic alcohol abuse. Uninterpretable (atypical) CDT patterns have been detected by both capillary electrophoresis (CE) and HPLC. The aim of this study was to evaluate the performance of HPLC as a second-line test for the interpretation of most frequent atypical CDT profiles detected by CE. METHODS: CDT was analyzed by CE (Capillarys 2, Sebia) on 9120 consecutive samples in a routine laboratory setting during a 2-year period. A commercial method (ClinRep CDT kit, Recipe) was employed to retest 123 (1.4%) samples with atypical CDT patterns on a Prominence LC-20AT HPLC (Shimadzu). RESULTS: CE-uninterpretable samples were categorized as having low transferrin (Tf) concentration (LT; n = 42, 0.5%), di-trisialotransferrin bridging (D-TB; n = 63, 0.7%) or atypical peak profile (APP; n = 18, 0.2%). CDT was detectable by HPLC in 58 of 123 (47%) samples including 21of 42 (50%) with LT, 27 of 63 (43%) with D-TB and 10 of 18 (56%) with APP. CONCLUSIONS: Second-line HPLC testing reduced uninterpretable samples by 47%, with similar rates of improvement regardless of the type of CDT pattern. The usefulness of HPLC as a second-line test for CDT should be evaluated according to cost-benefit considerations in the context of each laboratory.

Comparing the accuracy of PCR-capillary electrophoresis and cuticle microhistological analysis for assessing diet composition in ungulates: A case study with Pyrenean chamois.

The study of diet composition is required to understand the interactions between animal and plant ecosystems. Different non-invasive techniques applied on faecal samples have commonly been used for such purposes, with cuticle microhistological analysis (CMA) and emerging DNA-based methods being the most relevant. In this work, we refined and optimized a qualitative DNA-based approach combining PCR amplification of long trnL(UAA) and ITS2 fragments and capillary electrophoresis (PCR-CE), instead of short trnL(UAA) fragments and massive sequencing technologies commonly reported. To do so, we developed a controlled diet assay using a stabled Pyrenean chamois specimen (Rupicapra pyrenaica pyrenaica), which included representative herbaceous and shrubby plant species. We also assessed the impact of sample freshness on the diet determination of this mountain caprinae by exposing faecal samples to the outdoor environment for three weeks. Faecal samples from both experiments were analysed by qualitative PCR-CE and semi-quantitative CMA in order to compare the pros and cons of both approaches. Our results show that all of the offered plant species were detected by both methodologies although CMA over-detected shrubs compared to herbaceous species. At the same time, sample degradation due to sustained climate exposure is a limiting factor for molecular analysis, but not for CMA. Taken all together, our results suggest that the qualitative information obtained by CMA and PCR-CE can be interchangeable when faecal samples are fresh (less than one week after deposition) but, afterwards, molecular analysis underestimates diet composition probably due to DNA degradation. CMA, however, can accurately be used at least three weeks after defecation. Moreover, by combining the results of simultaneous PCR amplification of two complementary genes, this optimized PCR-CE methodology provides a reliable, feasible and more affordable alternative for multiple and routine analyses of complex samples. Neither CMA nor PCR-CE seems to solve comprehensively the quatification of herbivore diets and thus further research needs to be done.

CE-SDS method development, validation, and best practice-An overview.

CE-SDS has been implemented in the biopharmaceutical industry and is being used for the characterization of therapeutic proteins in most Biological License Applications currently submitted. An overview is presented on the separation mechanism, methodology, and good working practices/best practices. The CE-SDS platform method development and validation are discussed and typical scientifically and regulatory issues and troubleshooting situations are highlighted.

Evaluation of the suitability of the World Health Organization International Reference Reagent for Hb A2 quantitation (89/666) for continued use.

INTRODUCTION: The accurate determination of Hb A2 is a key marker when screening for a ß-thalassaemia carrier. Data from external quality assessment (EQA) exercises have shown a lack of alignment of Hb A2 quantitation both within and between methods. The only reference material available for Hb A2 quantitative assay at the time of writing is the World Health Organization International Reference Reagent (89/666; WHO IRR) prepared in the 1980s and not validated for all current methodologies. METHOD: The WHO IRR was analysed for Hb A2 concentration by 52 laboratories using a representative range of high-performance liquid chromatography and capillary electrophoresis analysers. The results of the analysis were compared to those of a whole blood EQA specimen of similar Hb A2 concentration, distributed in the same week. RESULTS: The mean Hb A2 value obtained for the WHO IRR was 5.17%, compared to the assigned value of 5.3%. The range of results returned was wide (4.0%-6.2%), with differences in the results observed by between and within analyser groups. A similar range of results was seen with the whole blood sample, although the bias observed between analyser types was different from that seen with the WHO IRR. CONCLUSION: The results may indicate a lack of commutability of the WHO IRR material, resulting from deterioration, matrix effects or changes in reagent formulation or calibration parameters. Further examination of the suitability of the WHO IRR (89/666) for continued use is required.

Development of a generic method for the determination of proton-pump inhibitors by capillary zoneelectrophoresis

A generic capillary zone electrophoresis method was developed for the analysis of four proton pump inhibitors: omeprazole, pantoprazole, lansoprazole and rabeprazole. During preliminary analysis screening of phosphate buffers at different pH levels was performed, in order to determine the optimum pH domain suitable for the simultaneous determination of all studied compounds. A face centered central composite design was employed for the optimization of separation conditions. The effect of buffer concentration, pH and applied voltage was studied; resolution between peaks and migration time of the last compound were considered as responses. Other factors as system temperature, injection parameters, capillary length, were held constant during the optimization process. The optimized conditions consisted of 40mM phosphate background electrolyte at pH 5.0, +25 kV applied voltage and 20 °C temperature. The migration order of the analytes was as follows: rabeprazole, omeprazole, lansoprazole and pantoprazole. Full resolution of all analytes was achieved within 9 minutes. The method was validated and proved to be suitable in terms of repeatability, sensitivity, linearity, accuracy and robustness. Determinations from commercially available pharmaceutical formulation were performed for omeprazole; good reproducibility and recovery were obtained.

Stability of reagents used for chiral amino acid analysis during spaceflight missions in high-radiation environments.

The search for biosignatures on spaceflight missions requires in situ instrumentation capable of highly selective and sensitive organic analyses. To this end, CE-LIF is a uniquely promising technique, capable of determining the type, abundance, and chirality of amino acids present in environmental samples at nanomolar concentrations. However, this type of assay requires several reagents that have not yet been used on spaceflight missions. A key concern, particularly for future missions to Europa, is the survivability of these critical components for CE separation and LIF detection under high levels of radiation. Here we present an investigation of the chemical stability of the reagents and associated fused silica capillary after a total ionizing dose of 300 krad, exceeding the predicted total ionizing dose for the potential Europa Lander Mission payload by two-fold. Neither the fused silica capillary nor the fluorescent dye (5-carboxyfluorescein succinimidyl ester) showed significant change in performance following irradiation. Following the irradiation of the pre-mixed background electrolyte, both migration time and resolution were affected. However, when the reagents (sodium tetraborate, sodium taurocholate, and γ-cyclodextrin) and the acetonitrile solution were irradiated separately and mixed afterwards, there was no change in the separation performance.

New HPLC instrument performance evaluation in HbA1c determination and comparison with capillary electrophoresis.

Glycated hemoglobin (HbA1c) measurement provides the most important medium to long-term marker of time-averaged glycemic status. Its relationship to clinical outcome in diabetes has been convincingly demonstrated for both type 1 and type 2 diabetes. The main HbA1c measurement methods for clinical routine are as follows: ion-exchange chromatography; affinity chromatography, capillary electrophoresis, immunoassay and enzymatic methods. In this study, we evaluated the analytical performances of a new HPLC instrument (Tosoh HLC-723 G11 in the VAR mode) in HbA1c analysis and compared it with a capillary electrophoresis instrument (Sebia Capillarys 2 Flex Piercing). HbA1c analysis was performed in parallel by both methods for 250 samples randomly chosen from healthy and diabetic subjects at 'Tor Vergata' University Hospital of Rome. Tosoh HLC-723 G11 showed good reproducibility for 10 days both in quality controls and in samples analyzed (%CV < 2%). We found good linearity for HbA1c values ranging from 15 mmol/mol (3.5%) to 178 mmol/mol (18.5%), with a correlation coefficient R2 = 1. In a comparison between Tosoh HLC-723 G11 and Capillarys 2FP a good correlation (r = 0.99) was found; however, Tosoh HLC-723 G11 showed higher values in the low range of HbA1c and lower in the high range (Tosoh HLC-723 G11 = 4.3043 + 0.913 Capillarys 2FP; p < 0.001). Tosoh HLC-723 G11 showed good repeatability, reproducibility, accuracy and automated simplicity, and it seemed suitable for routine use in clinical chemistry laboratories.

Interlaboratory method validation of icIEF methodology for analysis of monoclonal antibodies.

CE is central to the analysis, process development and approval of therapeutic monoclonal antibodies (mAbs). Recently, imaged capillary isoelectric focusing (icIEF) has emerged as a powerful technique for quantitative protein charge heterogeneity monitoring and characterization, particularly for mAbs. However, icIEF has yet to be validated for therapeutically relevant mAbs adhering to the ICH guideline (International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use). Here, for the first time, icIEF technology was validated by 10 laboratories across 8 independent companies using a therapeutic mAb. The parameters of this method validation strictly follow the guideline of the ICH. This guideline includes specificity, precision, accuracy, linearity, range, LOQ and robustness. These results represent a significant step forward in standardizing the use of icIEF methods for the clinical approval of therapeutic mAbs.

Electromembrane extraction and capillary electrophoresis with capacitively coupled contactless conductivity detection: Multi-extraction capabilities to analyses trace metals from saline samples.

Simultaneous electromembrane extraction (EME) of six trace metal cations (Cu2+ , Zn2+ , Co2+ , Ni2+ , Pb2+ , Cd2+ ) from saline samples was investigated. CE with capacitively coupled contactless conductivity detection (C4 D) was used to determine the metals in acceptor solutions due to its excellent compatibility with the minute volumes of acceptor solutions. Bis(2-ethylhexyl)phosphate (DEHPA) was selected as a suitable nonselective modifier for EME transport of target metal cations. Both, the individual effect of each major inorganic cation (Na+ , K+ , Ca2+ , Mg2+ ) and their synergistic effect on EME of the trace metal cations were evaluated. In both cases, a decrease in extraction efficiency was observed when major inorganic cations were present in the sample. This effect was more significant for Ca2+ and Mg2+ . The system was optimized for simultaneous extractions of the six target metals from saline samples (50 mM Na+ , 5 mM Mg2+ , 1 mM K+ , and 1 mM Ca2+ ) and following EME conditions were applied. Organic phase consisted of 1-nonanol containing 1% (v/v) DEHPA, acceptor solution was 1 M acetic acid (HAc) and sample pH was adjusted to 5. Sample was stirred at 750 rpm and EMEs were carried out at extraction potential of 10 V for 20 min. The method presented a repeatability between 8 and 21.8% (n = 5), good linearity in 0.5-10 µM concentration range (R2  = 0.987-0.999) and LOD better than 2.6 nM. Applicability of the EME-CE-C4 D method to the analyses of metal cations in drinking water, seawater, and urine samples was also demonstrated.