Dual-probe ligation without PCR for fluorescent sandwich assay of EGFR nucleotide variants in magnetic gene capture platform.

A simple, rapid, and highly efficient fluorescent detection technique without PCR through dual-probe ligation with the genetic capture of magnetic beads and reported probe was developed for determination of epidermal growth factor receptor (EGFR) gene exon 19 deletions. The EGFR exon 19 deletion mutation makes up 48% of all mutations associated with anti-tyrosine kinase inhibition sensitivity, and thus, the EGFR nucleotide variant is very important in clinical diagnosis. In this approach, the dual-probe ligation was designed to target exon 19 deletion. The magnetic genetic captured system was then applied to capture the successful dual-probe ligation. Thereafter, a reporter probe which is coupled with 6-fluorescein amidite (6-FAM) was introduced to hybridize with dual-probe ligation product on the surface of streptavidin magnetic beads, and finally, the supernatant was taken for fluorescence measurements for distinguishing mutant types from wild types. After optimization (the RSD of the fluorescent intensity was less than 4.5% (n = 3) under the optimal condition), 20 blind DNA samples from the population were analyzed by this technique and further confirmed by direct sequencing. The results of our assay matched to those from direct sequencing data, evidencing that the developed method is accurate and successful. These 20 blind DNA samples were diagnosed as wild and then spiked with different percentages of the mutant gene to quantify the ratio of the wild and mutant genes. This strategy was also successfully applied to quantify the ratio of the wild and mutant genes with good linearity at the λex/λem of 480 nm/520 nm (r = 0.996), and the limit of detection reached 1.0% mutant type. This simple fluorescent detection of nucleotide variants shows its potential to be considered a tool in biological and clinical diagnosis. Importantly, this strategy offers a universal detection capability for any kind of mutation (point, deletion, insertion, or substitution) in a gene of interest.

One Single Tube Reaction of Aptasensor-Based Magnetic Sensing System for Selective Fluorescent Detection of VEGF in Plasma.

In this study, a simple, easy and convenient fluorescent sensing system for the detection of the vascular endothelial growth factor (VEGF) based on VEGF aptamers, aptamer-complementary fluorescence-labeled probe and streptavidin magnetic beads was developed in one single tube. The VEGF is the most important biomarker in cancer, and it is investigated that the serum VEGF level varied according to the different types and courses of cancers. Hence, efficient quantification of VEGF is able to improve the accuracy of cancer diagnoses and the precision of disease surveillance. In this research, the VEGF aptamer was designed to be able to bind with the VEGF by forming G-quadruplex secondary structures; then, the magnetic beads would capture the non-binding aptamers due to non-steric interference; and finally, the fluorescence-labeled probes were hybridized with the aptamers captured by the magnetic beads. Therefore, the fluorescent intensity in the supernatant would specifically reflect the present VEGF. After an overall optimization, the optimal conditions for the detection of VEGF were as followed, KCl, 50 µM; pH 7.0; aptamer, 0.1 µM; and magnetic beads, 10 µL (4 µg/µL). The VEGF could be well quantified within a range of 0.2-2.0 ng/mL in plasma, and the calibration curve possessed a good linearity (y = 1.0391x + 0.5471, r = 0.998). The detection limit (LOD) was calculated to be 0.0445 ng/mL according to the formula (LOD = 3.3 × σ/S). The specificity of this method was also investigated under the appearance of many other serum proteins, and the data showed good specificity in this aptasensor-based magnetic sensing system. This strategy provided a simple, sensitive and selective biosensing platform for the detection of serum VEGF. Finally, it was expected that this detection technique can be used to promote more clinical applications.

Copper nanoclusters on specific-primer PCR fragments with magnetic capture for the label-free fluorescent sensing of the T315I single nucleotide variant in the BCR-ABL1 gene.

In this study, a simple and facile procedure using the all or none formation of double-stranded DNA-templated copper nanoclusters on specific-primer PCR fragments was designed to fluorescently identify the T315I single nucleotide variant on the BCR-ABL1 gene. Chronic myeloid leukaemia (CML), a disease caused by the BCR-ABL1 fusion of tyrosine kinase, is well known for the T315I mutation that causes tyrosine kinase inhibitors (TKIs) to be resisted due to the alternative structure of the drug-binding site. Therefore, it is an important single nucleotide variant for clinical detection. In this study, only specific functional primers and the digestion of the wild genotype from the T315I mutation site with specific restriction enzymes were designed, and the different digested products could then be captured using magnetic beads. The final products would allow for fluorescent sensing via the all or none formation of double-stranded DNA-templated copper nanoclusters for the detection of the T315I mutation. This study has been successfully applied for identifying wild and mutant homozygotes and the mutant/wild heterozygote of the T315I mutation. It is expected that this analytical system can serve as a tool for the clinical diagnosis of T315I mutations and be applied to real samples of CML patients in the future.

A chemometric experimental design with three-step stacking capillary electrophoresis for analysis of five tobacco-specific nitrosamines in cigarette products.

Tobacco-specific nitrosamines (TSNAs) as carcinogens endanger our health and life from cigarette products. However, the safe range of TSNAs levels in commercial cigarette products has not yet been established. For the purpose of safety and supervision, a three-step stacking approach including field amplified sample injection (FASI), sweeping, and analyte focusing by micelle collapse (AFMC), was developed for the simultaneous determination of five TSNAs levels in cigarette products. This approach also involved aspects of chemometric experimental design, including fractional factorial design and central composite design. After the multilevel optimization of the experimental design, the five TSNAs were well separated. The LOD (S/N = 3) values of the N´-nitrosonornicotine (NNN), N´-nitrosoanatabine (NAT), N´-nitrosoanabasine (NAB), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in the FASI-sweeping-AFMC CE approach were 1.000 ng/mL, 0.500 ng/mL, 0.125 ng/mL, 1.000 ng/mL, and 0.500 ng/mL respectively. The results of relative standard deviation (RSD) and relative error (RE) were all less than 3.35%, demonstrating good precision and accuracy. Finally, this novel approach was further applied to monitor three commercial cigarette products, and a range of 250.1-336.6 ng/g for NNN, 481.6-526.7 ng/g for NAT, 82.2-247.6 ng/g for NAB, 167.7-473.7 ng/g for NNAL, and 39.4-246.7 ng/g for NNK could be observed among these. Based on these results, the novel CE stacking strategy was successfully applied for the analysis of five TSNAs levels in cigarette products and could serve as a tool for assays of quality control of nitrosamines.

A three-step stacking capillary electrophoresis of field-amplified sample injection, sweeping, and micellar collapse for determination of dabigatran and its active metabolite in human plasma.

A three-step stacking capillary electrophoresis (CE) composed of field-amplified sample injection, sweeping, and analyte focusing by micellar collapse (FASI-sweeping-AFMC) was developed to determine dabigatran (D) and its major active metabolite, dabigatran acyl-beta-d-glucuronide (DAG), in human plasma. After optimization and validation, this novel approach was further applied to monitor 5 real samples, and the 25.2-186.8 ng mL-1 D could be observed among those. Based on these results, the novel CE stacking strategy was successfully applied for the analysis of D and DAG in human plasma and could be served as a tool for clinical assays.

Identifiable universal fluorescent multiplex PCR equipped with capillary electrophoresis for genotyping of exons 1 to 5 in human red and green pigment genes.

Congenital red and green color blindness is the most X-linked recessive disorder in humans caused by deletions or gross structural rearrangements of the visual pigment gene array that lead to altered the functions of visual pigments in their retina differ from normal. The incidence is about 7-10% in male and close association of X-linked recessive disorders (such as: hemophilia A, hemophilia B, duchenne muscular dystrophy). However, the traditional genetic analysis methods are time-consuming and low-efficiencies. Therefore, the purpose of the study is to develop a rapid method for genotyping of red and green pigment genes. We describe herein the first method for simultaneous evaluation of ten exons in the red and green pigment genes for genetic analysis. A forward specific primers with identifiable universal fluorescent multiplex PCR (FSIUFM-PCR) method utilized one universal primer (containing two universal non-human sequences) and forward specific primers in the multiplex PCR reaction system for simultaneously fluorescent labeling of eleven gene fragments (ten exons in red and green pigment genes and one internal standard). All the PCR products were analyzed on capillary electrophoresis with short-end injection, which had the advantage of high resolution and rapid separation. Of all 80 detected individuals, 7 subjects with color vision deficiencies (including 3 subjects only had red exons 1-5, 4 subjects had a specific red-green or green-red hybrid gene and 73 subjects with normal color vision). All genotyping results showed good agreement with DNA sequencing data. This method provided a better potential technique for genotyping and identifying of red and green pigment genes. In addition, FSIUFM-PCR method will be useful in many fields, such as diagnosis of diseases, analysis of polymorphisms and quantitative assay.

Stable Luminescent Poly(Allylaminehydrochloride)-Templated Copper Nanoclusters for Selectively Turn-Off Sensing of Deferasirox in ß-Thalassemia Plasma.

Highly stable and facile one-pot copper nanoclusters (Cu NCs) coated with poly(allylamine hydrochloride) (PAH) have been synthesized for selectively sensing deferasirox (DFX) in ß-thalassemia plasma. DFX is an important drug used for treating iron overloading in ß-thalassemia, but needs to be monitored due to certain toxicity. In this study, the PAH-Cu NCs showed highly stable fluorescence with emission wavelengths at 450 nm. The DFX specifically interacted with the copper nanocluster to turn off the fluorescence of the PAH-Cu NCs, and could be selectively quantified through the fluorescence quenching effect. The linear range of DFX in plasma analyzed by PAH-Cu NCs was 1.0-100.0 µg/mL (r = 0.985). The relative standard deviation (RSD) and relative error (RE) were lower than 6.51% and 7.57%, respectively, showing excellent reproducibility of PAH-Cu NCs for sensing DFX in plasma. This method was also successfully applied for an analysis of three clinical plasma samples from ß-thalassemia patients taking DFX. The data presented high similarity with that obtained through a capillary electrophoresis method. According to the results, the PAH-Cu NCs could be used as a tool for clinically sensing DFX in human plasma for clinical surveys.

Molecular inversion probe-rolling circle amplification with single-strand poly-T luminescent copper nanoclusters for fluorescent detection of single-nucleotide variant of SMN gene in diagnosis of spinal muscular atrophy.

In this study, a simple fluorescent detection of survival motor neuron gene (SMN) in diagnosis of spinal muscular atrophy (SMA) based on nucleic acid amplification test and the poly-T luminescent copper nanoclusters (CuNCs) was established. SMA is a severely genetic diseases to cause infant death in clinical, and detection of SMN gene is a powerful tool for pre- and postnatal diagnosis of this disease. This study utilized the molecular inversion probe for recognition of nucleotide variant between SMN1 (c.840 C) and SMN2 (c.840 C  >  T) genes, and rolling circle amplification with a universal primer for production of poly-T single-strand DNA. Finally, the fluorescent CuNCs were formed on the poly-T single-strand DNA template with addition of CuSO4 and sodium ascorbate. The fluorescence of CuNCs was only detected in the samples with the presence of SMN1 gene controlling the disease of SMA. After optimization of experimental conditions, this highly efficient method was performed under 50 °C for DNA ligation temperature by using 2U Ampligase, 3 h for rolling circle amplification, and the formation of the CuNCs by mixing 500 µM Cu2+ and 4 mM sodium ascorbate. Additionally, this highly efficient method was successfully applied for 65 clinical DNA samples, including 4 SMA patients, 4 carriers and 57 wild individuals. This label-free detection strategy has the own potential to not only be a general method for detection of SMN1 gene in diagnosis of SMA disease, but also served as a tool for detection of other single nucleotide polymorphisms or nucleotide variants in genetic analysis through designing the different sensing probes.

The matrix of SDS integrated with linear hydrophilic polymer for resolution of high- and low-molecular weight hyaluronic acids in MEKC.

Hyaluronic acid (HA), a multi-functional material, has a high dispersion in molecular weight, and the functions of HA are determined through the size. Nevertheless, hyaluronic acid mixtures are not easily separated due to their polydispersity. In this study, a capillary electrophoresis strategy was developed for resolution of different molecular-weight HA without enzymatic digestion. Here, hyaluronic acid mixtures with low molecular weight (380 kD; LHA) and high molecular weight (2180 kD; HHA) were successfully resolved by the SDS integrated with low molecular-weight polymer in capillary electrophoresis. By optimizing experimental conditions, the separation of LHA and HHA was completed within 14 min. The optimal conditions were as follows: the running buffer was 25 mM borate buffer (pH 9.75) containing 30 mM SDS and 10% polyethylene glycol (MW: 8000); applied voltage was 20 kV (detector at cathode side) and separation temperature was set at 25 °C. The data of method validation showed that calibration plots were linear (r ≥ 0.9977) over a range of 10-50 µg/mL for LHA, and 40-200 µg/mL for HHA. In the evaluation of precision and accuracy for this method, the RSD and RE values were all less than 4.2%. This fascinating technique was successfully applied to the quality control of cosmetic and pharmaceutical containing different ratios of LHA and HHA, and it was feasible for serving as a tool to quantitatively analyze different sizes of HA for clinical survey.

Specific Gene Capture Combined with Restriction-Fragment Release for Directly Fluorescent Genotyping of Single-Nucleotide Polymorphisms in Diagnosing Spinal Muscular Atrophy.

In this study, a fast and simple fluorescent genotyping strategy, streptavidin magnetic beads combined with biotin-coupled PCR and restriction-fragment release, was developed for determination of nucleotide variants. This method was further applied for analyzing SMN1 gene in diagnosis of spinal muscular atrophy (SMA). After biotin-coupled PCR, the streptavidin magnetic beads would capture the biotin-labeled SMN genetic fragments, and then the restriction enzyme of HPY188I could only digest and release the fluorescent end of SMN1 genetic fragment into the supernatant. Therefore, the SMN1 gene could be easily fluorescently quantified, and SMN2 would not, for diagnosis of SMA. The copy number of the SMN1 gene could be regressed using the relative fluorescent unit versus the known copy number, and the coefficient of correlation is equal to 0.9617 ( r = 0.9617). In this research, a total of 16 blind DNA samples were analyzed, including 6 wild types, 5 carriers, and 5 SMA patients. Importantly, this fast, simple, and highly efficient method is universal for detection of all nucleotides variants by replacing the specific restriction enzyme. This technique has the potency to be served as a tool for fast and accurate diagnosis of genotypes in clinical medicine.

Erratum: Removal of Diethylhexyl Phthalate from Hands by Handwashing: Evidence from Experimental N-of-1 and Crossover Designs.

A correction to this article has been published and is linked from the HTML version of this paper. The error has been fixed in the paper.

Molecular inversion probes equipped with discontinuous rolling cycle amplification for targeting nucleotide variants: Determining SMN1 and SMN2 genes in diagnosis of spinal muscular atrophy.

The novel techniques of molecular inversion probes (MIPs) combined with discontinuous rolling cycle amplification (DRCA) was developed for determination of the multi-nucleotide variants at single base. The different-length MIPs, a padlock-probe based technology, are designed to simultaneously recognize the identical nucleotide variants. After ligation and DRCA, the different-length genetic products representing the certain genotypes could be simply determined by the short-end capillary electrophoresis (CE) method. By using MIPs-DRCA method, the various gene dosages of SMN1 and SMN2 genes in homologous or heterologous subjects were successfully quantified for diagnosis of spinal muscular atrophy (SMA). The length of the MIP for SMN1 gene was 106 bp, and for SMN2 gene was 86 bp. After method optimization, the MIP products of SMN1 and SMN2 were well separated with the resolution of 1.13 ± 0.17 (n = 3) within 10 min. There were total of 56 DNA blind samples analyzed by this strategy, including 38 wild types, 12 carriers and 6 SMA patients, and the data of gene dosages was corresponding to those analyzed by conformation sensitive CE and denatured high performance liquid chromatography (DHPLC) methods. This MIPs-DRCA method which could be applied to simultaneously genotype multi nucleotide variants at single base, such as K-ras gene, was very feasible for determination of genetic diseases in clinical.

Dialkyl anionic surfactant in field-amplified sample injection and sweeping-micellar electrokinetic chromatography for determination of eight leanness-promoting ß-agonists in animal feeds.

The beta-adrenergic agonists (ß-agonists) working as repartitioning agents that make the carcass leaner and enhance the feeding efficiency in animals have been banned in the European Union, China and Taiwan. Here, traditional anionic surfactants, such as sodium dodecyl sulfate (SDS) were replaced with sodium di-(2-ethylhexyl)-sulfosuccinate (AOT) in field-amplified sample injection and sweeping-micellar electrokinetic chromatography (FASI-sweeping MEKC) for simultaneous analysis of eight ß-agonists in animal feeds. The AOT vesicles provided a better resolution of ß-agonists than micelles of SDS. The detection limits of the eight ß-agonists were above 5ng/mL by using this stacking capillary electrophoresis (CE) method. In comparison of traditional MEKC method (sample injection, 1psi for 5s), the stacking strategy provided 400-2000 fold sensitivity enhancement. After method validation, this method was successfully applied for analyzing four animal feeds, and none ß-agonist was detected. This strategy possessing good resolution of eight ß-agonists was suitable for serving as a tool for routine analysis of animal feeds.

Removal of Diethylhexyl Phthalate from Hands by Handwashing: Evidence from Experimental N-of-1 and Crossover Designs.

Phthalate exposure through skin is often neglected due to the small quantity and limited dermal absorption rate. However, free phthalate can be ingested by hand-to-mouth action or by contact with food. To evaluate the effectiveness in removing phthalate exposure on hand, we compare here the removal efficiency of di-(2-ethylhexyl)phthalate (DEHP) on hands by handwashing with soap-and-water versus water-only. In two three-day N-of-1 trials, residual DEHP was measured in a single female adult who washed exposed hands with soap-and-water or water-only. Subsequently, a crossover study was performed by randomly assigning another 28 subjects equally to wash with soap-and-water or with water-only, and then each one received the other treatment 24 hrs later. In the N-of-1 trials, mean DEHP removal rates range from 95.9% (SD = 0.1%) to 97.0% (SD = 2.5%) for soap-and-water handwashes, and 1.8% (SD = 0.1%) to 7.0% (SD = 0.3%) (n = 3) for water-only. In the crossover study, mean removal rate was 94.6% (SD = 6.5%) for handwashing with soap-and-water (n = 28) and 8.7% (SD = 5.7%) for water-only (n = 28). We concluded that handwashing with soap-and-water removes 80% more DEHP than handwashing with water alone, and may be a cost-effective way of removing other endocrine disruptors from hands.

Determination of deferasirox in human plasma by short-end injection and sweeping with a field-amplified sample stacking and micellar electrokinetic chromatography.

A field-amplified sample stacking-sweeping micellar electrokinetic chromatography with short-end injection was established for determination of deferasirox (DFX) in plasma. DFX was extracted from plasma and reconstituted with deionized water (lower conductivity solution). Capillary (effective length, 10cm) was filled with background electrolyte (40mM phosphate buffer, pH 4.5, containing 20% methanol). After sample loading from outlet end at 5psi for 15s, separation was carried out by applying high voltage at 15kV for 10min. Sodium dodecyl sulfate (SDS) was used to sweep DFX for enhancing sensitivity. The optimal CE separation conditions were 40mM phosphate buffer at pH 4.5 containing 100mM SDS and 20% methanol. The analysis time was about 3.5min for DFX. The calibration curve of DFX was ranged from 1 to 20µg/ml. The linearity (r) was more than 0.998. RSD and RE in intra- and inter-day assays were all below 12.14%. The limit of detection (LOD, S/N=3) for DFX was 0.3µg/ml. The sensitivity enhancement factor between sweeping-FASS MEKC and capillary zone electrophoresis is 3.3. Finally, the method was applied for determination of DFX in ß-thalassemia patients.

Therapeutic deferoxamine and deferiprone monitoring in ß-thalassemia patients' plasma by field-amplified sample injection and sweeping in capillary electrophoresis.

One CE method was established for detecting deferoxamine (DFO) and deferiprone (DFR) in plasma. For ß-thalassemia patients, DFO and DFR are major medicines to treat the iron overload caused by blood transfusion. Field-amplified sample injection combined with sweeping was used for sensitivity enhancement in CE. This method was performed on an uncoated fused-silica capillary. After liquid-liquid extraction, the plasma samples were electrokinetically injected into capillary at +10 kV for 180 s. The phosphate buffer (100 mM) containing 50 mM triethanolamine was used as the BGE (pH 6.6). Separation buffer was phosphate buffer (100 mM, pH 3.0) containing 150 mM SDS. This method showed good linearity (r ≥ 0.9960). Precision and accuracy were evaluated by the results of RSD and relative error of intrabatch and interbatch analyses, and all of the absolute values were less than 6.12%. The LODs (S/N = 3) were 200 ng/mL for DFO, and 25 ng/mL for DFR. The LOQ (S/N = 10) of DFO and DFR were 600 and 75 ng/mL, respectively. This method was applied for clinical applications of five ß-thalassemia patients.

High-Performance Liquid Chromatographic Analysis of Sarcosine as a Fluorescent Levofloxacin Derivative.

Sarcosine, N-methyl glycine, could be used as a biomarker for the diagnosis of prostate cancer. It exists in biosamples at low levels; therefore, sensitive methods are necessary for its detection. In this study, we developed a sensitive and selective method for the analysis of sarcosine, based on derivatizing sarcosine with a fluorescent reagent levofloxacin acyl chloride. The resulting derivative is highly responsive to a fluorimetric detector (λex = 290 nm, λem = 460 nm). The sarcosine derivative can be separated from its molecular isomers (α-l-alanine, α-d-alanine and ß-alanine) on a hexyl-phenyl column by gradient elution using sodium acetate buffer (pH 3.8; 50 mM) and tetrahydrofuran as the mobile phase. The method showed a determination range of sarcosine in water over 44.5-1780.0 ng/mL (0.5-20.0 µM) and the limit of detection at 8.9 ng/mL (0.1 µM) (S/N = 3 with 20 µL injected). Intra- and inter-day precision (as % relative standard deviation) and accuracy (as % relative error) were all below 4.8%. Application of the method to the analysis of sarcosine in human urine proved feasible.

Effect of Di(2-ethylhexyl)phthalate on Helicobacter pylori-Induced Apoptosis in AGS Cells.

Plastic products are wildly used in human life. Di(2-ethylhexyl)phthalate (DEHP) is an essential additive in plastic manufacturing and is used as plasticizer for many products including plastic food packaging. DEHP is a teratogenic compound and can cause potent reproductive toxicity. DEHP can also cause liver damage, peroxisome proliferation, and carcinogenesis. DEHP is also strongly associated with peptic ulcers and gastric cancer; however, the underlying effect and mechanism of DEHP on the gastrointestinal tract are not entirely clear. The oral infection route of H. pylori parallels the major ingestion route of DEHP into the human body. Therefore, we wanted to study the effect of DEHP and H. pylori exposure on the human gastric epithelial cell line, AGS (gastric adenocarcinoma). The viability of the AGS cell line was significantly lower in 80 µ M-DEHP and H. pylori (MOI = 100 : 1) coexposure than DEHP or H. pylori alone. DEHP and H. pylori coexposure also induced caspase-3 activation, and increased Bax/Bcl-2 ratio and DNA fragmentation in AGS cells. These results indicate that DEHP can enhance H. pylori cytotoxicity and induce gastric epithelial cell apoptosis. Therefore, it is possible that DEHP and H. pylori coexposure might enhance the disruption of the gastric mucosa integrity and potentially promote the pathogenesis of gastric carcinogenesis.

Membrane sampling with microdialysis coupled to HPLC/UV for on-line simultaneous determination of melamine and cyanuric acid in non-dairy coffee creamer.

An on-line microdialysis/high-performance liquid chromatography method was developed for the simultaneous determination of melamine and cyanuric acid in non-dairy coffee creamer. To collect these analytes from aqueous samples, the microdialysis system featured a microdialysis probe incorporating a polyarylethersulfone membrane and employed 0.05 M HCl in 0.1% (v/v) MeOH as the perfusate, with optimal efficiency obtained at a flow rate of 1 µL min(-1). The chromatographic conditions were optimized when using a reverse-phase phenyl column and a mobile phase of phosphate buffer solution in 10% (v/v) MeOH, buffered at pH 3.0. Good linearity relationship (r(2) > 0.9987), intra- and inter-day precisions (RSDs < 6.6%), recoveries (96.9 - 105.0%), and limits of detection (melamine, 3 ppb; cyanuric acid, 150 ppb) were observed for the two analytes. This method has been successfully applied to simultaneous determination of melamine and cyanuric acid in commercial creamers with the recoveries in the range of 97.5 to 102.6%.

Using an on-line microdialysis/HPLC system for the simultaneous determination of melamine and cyanuric acid in non-dairy creamer.

The recent revelation of melamine (MEL) contamination in foodstuffs in China has rocked the international public health community. Many food categories have been involved in this scandal, including non-dairy creamer (NDC). In this study, we investigated the use of hollow-fiber microdialysis (MD) sampling coupled on-line with high-performance liquid chromatography (HPLC) as an alternative to sample pretreatment for the direct determination of MEL and its analogue cyanuric acid (CYA) in NDC. After MD sampling, the dialysate was injected on-line into the chromatographic system for analysis of MEL and CYA with UV detection at 203 nm. We monitored the effects of various parameters affecting the MD efficiency, namely the characteristics of the MD probe membrane, the flow-rate and the nature of the polarity modifier in the perfusion stream, and the addition of salt in the sample solution. The optimal enrichment efficiency for collecting MEL and CYA from aqueous NDC samples occurred with MD sampling using a hollow polysulfone MD fiber and MeOH as the perfusate at a flow rate of 10 µL min(-1). The optimized chromatographic conditions involved using a reversed-phase phenyl column and a mobile phase of 5 mM phosphate buffer in 10% (v/v) MeOH, buffered at pH 6.5. Detection was linear in the concentration range from 0.02 to 5 ppm for MEL and from 2 to 100 ppm for CYA, with detection limits of 1 ppb for MEL and 30 ppb for CYA. The volume of perfusate required to extract MEL and CYA from the NDC solution was only 21 µL. The total MD sampling time was 2.1 min. This method allows the sensitive, eco-friendly, and rapid determination of MEL and CYA in NDC-a risk food for economically motivated adulteration.