Chun-Chi Wang

Large volume sample stacking with EOF and sweeping in CE for determination of common preservatives in cosmetic products by chemometric experimental design

This study proposes a capillary electrophoresis method incorporating large volume sample stacking, EOF and sweeping for detection of common preservatives used in cosmetic products. The method was developed using chemometric experimental design (fractional factorial design and central composite design) to determine multiple separation variables by efficient steps. The samples were loaded by hydrodynamic injection (10 psi, 90 s), and separated by phosphate buffer (50 mM, pH 3) containing 30% methanol and 80 mM SDS at −20 kV. During method validation, calibration curves were found to be linear over a range of 5–100 μg/mL for butyl paraben and isobutyl paraben; 0.05–10 μg/mL for ethyl paraben; 0.2–50 μg/mL for dehydroacetic acid; 0.5–70 μg/mL for methyl paraben; 5–350 μg/mL for sorbic acid; 0.02–450 μg/mL for p‐hydroxybenzoic acid and 0.05–10 μg/mL for salicylic acid and benzoic acid. The analytes were analysed simultaneously and their detection limits (S/N = 3) were down to 0.005–2 μg/mL. The analysis method was successfully used for detection of preservatives used in commercial cosmetics.

A novel stacking method of repetitive large volume sample injection and sweeping MEKC for determination of androgenic steroids in urine

In this research, a novel stacking capillary electrophoresis method, repetitive large volume sample injection and sweeping MEKC (rLVSI-sweeping MEKC) were developed to analyze the presence of three androgenic steroids considered as sport doping drugs, testosterone (T), epitestosterone (E) and epitestosterone glucuronide (EG) in urine. This method provides better sensitivity enhancement than the traditional large volume sample stacking-sweeping strategies due to sensitivity enhancement by repetitive injections. This multiple sampling method enhances sensitivity of monitoring of urine samples by UV detection (254 nm). Firstly, the phosphate buffer was filled into an uncoated fused silica capillary and the samples were injected into the capillary at 10 psi for 20s, and then stacked at -10 kV for 1 min using phosphate buffer containing SDS. The above injecting and stacking steps were repeated five times. Finally, separation was performed at -20 kV, using phosphate buffer containing methanol, SDS and (2-hydroxypropyl)-β-cyclodextrin. Method validation showed that calibration plots were linear (r≥0.997) over a range of 5-200 ng mL(-1) for T, 20-200 ng mL(-1) for E and 0.5-500 ng mL(-1) for EG. The limits of detection were 1.0 ng mL(-1) for T, 5.0 ng mL(-1) for E and 200.0 pg mL(-1) for EG. When evaluating precision and accuracy, values of RSD and RE in intra-day (n=3) and inter-day (n=5) analysis were found to be less than 10.0%. Compared with the simple LVSS-sweeping, which is also a stacking strategy, this method further improves sensitivity up to 25 folds (~2500 folds with MEKC without preconcentration). This method was applied to monitor 10 athletes urine, and did not detect any analyte. The novel stacking method was feasible for monitoring of doping by sportsmen.

Analysis of ten abused drugs in urine by large volume sample stacking–sweeping capillary electrophoresis with an experimental design strategy

A statistical tool equipped with Plackett-Burman design (PBD) and central composite design (CCD) was used for fast stacking analysis of ten frequently consumed drugs, namely codeine, morphine, methamphetamine, ketamine, alprazolam, clonazepam, diazepam, flunitrazepam, nitrazepam and oxazepam, by capillary electrophoresis (CE). This statistical design is expected to help quick analysis with few procedures, avoiding tedious work required because of the large number of variables or parameters. A large volume sample stacking (LVSS)-sweeping CE is developed for concentrating and analyzing the 10 abused drugs. First, phosphate buffer (50 mM, pH 2.3) containing methanol was filled into a capillary and then the extracted urine sample was loaded (1 psi, 200 s) to enhance sensitivity. The sweeping and separating steps were completed simultaneously by phosphate buffer (50 mM, pH 2.3) containing methanol and sodium dodecyl sulfate, within 15 min. Better resolution was obtained by the experimental design than the one factor at a time (OFAT) approach. During method validation, calibration plots were linear (r>0.998), over a range of 25-1500 ng/mL for the six benzodiazepines, methamphetamine and ketamine, and 50-3000 ng/mL for codeine and morphine. The RSD of precision and absolute RE of accuracy in intra-day and inter-day assays were below 14.54% and 16.61%, respectively. The minimum limits for detection (S/N=3) of analytes were in the range of 7.5-30 ng/mL. This stacking method increased sensitivity more than 200-fold and can be applied for detection of the presence of methamphetamine in an abusers urine (3600 ng/mL), which was confirmed by GC-MS. The method is considered feasible for fast screening of abused drugs in urine.

Quantification of SMN1 and SMN2 genes by capillary electrophoresis for diagnosis of spinal muscular atrophy

We present the first CE method for the separation and quantification of SMN1 and SMN2 genes. Spinal muscular atrophy (SMA) is an inherited neuromuscular disorder deleted or mutated in SMN1 gene and retained at least one copy of SMN2 gene. However, these two genes are highly homologous, differentiation and quantification of SMN1 and SMN2 are therefore required in diagnosis to identify SMA patients and carriers. We developed a fluorescence‐labeled conformation‐sensitive CE method to quantitatively analyze PCR products covering the variable position in the SMN1/SMN2 genes using a copolymer solution composed of hydroxyethylcellulose and hydroxypropylcellulose. The DNA samples included 24 SMA patients, 52 parents of SMA patients (obligatory carriers), and 255 controls. Those 331 samples were blind analyzed to evaluate the method, and the results compared with those obtained using denaturing HPLC (DHPLC). Validation of accuracy was performed by comparing the results with those of DHPLC. Nine of total samples showed different results. Diagnosis of one fetus DNA among them was related to abortion or not, which was further confirmed by gel electrophoresis and DNA sequencing. Our method showed good coincidence with them, and proved the misdiagnosis of DHPLC. This simple and reliable CE method is a powerful tool for clinical genotyping of large populations to detect carriers and SMA patients.

Enantioseparation of (±)-threo-methylphenidate in human plasma by cyclodextrin-modified sample stacking capillary electrophoresis

The (±)-threo-methylphenidate ((±)-threo-MP) is widely used for treatment of attention-deficit hyperactivity disorder (ADHD). According to clinical evidence, (+)-threo-MP possesses higher potency than (-)-threo-MP. Due to ppb level in plasma, till now, none of the capillary electrophoresis (CE) methods have been able to provide adequate sensitivity for therapeutic (±)-threo-MP monitoring. In this study, a cyclodextrin-modified field-amplified sample stacking CE method (CD-FASS-CE) for enantioseparation of (±)-threo-MP in human plasma was established for clinical applications. Phosphate buffer (50mM, pH 3.0) was filled into uncoated fused silica capillary (40 cm, 50 μm I.D.), followed by a water plug (0.5 psi, 10s). Electrokinetic injection (6 kV, 200 s) was used to load samples and to enhance sensitivity. Stacking and separation were performed at 20 kV and 200 nm using phosphate buffer (50mM, pH 3.0) containing 20mM HP-β-CD and 30 mM triethanolamine. Analytes were separated simultaneously by using CD-FASS-CE and had a lower detection limit of equal to the sub-ppb level. Linear calibration curves were obtained from 1 to 80 ng/mL (r=0.998). The limit of detection for both isomers was 600 pg/mL. RSD and RE of precision and accuracy in intra- and inter-day assays were below 7.89%. This method was further applied to analyze (±)-threo-MP in four healthy Asian volunteers and that provided some relevant information for clinical treatments.

Label-Free Fluorescent Copper Nanoclusters for Genotyping of Deletion and Duplication of Duchenne Muscular Dystrophy

Real applications in clinical diagnosis of label-free fluorescent copper nanoclusters (CuNCs) are demonstrated. Double-strand DNA (dsDNA) can act as an effective template for the formation of CuNCs, which can be used to distinguish the deletion or duplication genotypes of Duchenne muscular dystrophy (DMD) due to different fluorescent intensities. After PCR, the DMD amplicons reacted with copper ion by reduction of ascorbic acid and generated fluorescence. The exons of the DMD gene were taken as the model analytes for genetic diagnosis. In this sensing system, the deletion type does not show fluorescence; on the other hand, the duplication type emits higher fluorescence than normal type. Parameters of this sensing system were optimized, including PCR conditions, levels of copper ion and ascorbate, and reaction time. The DMD-dominated exons 45, 46, and 47 were detected, and the method was applied to six samples of DMD patients. The results were consistent with those of the multiplex ligation-dependent probe amplification method. This strategy was feasible to detect all exons of this disease.

Universal multiplex PCR and CE for quantification of SMN1/SMN2 genes in spinal muscular atrophy

We established a universal multiplex PCR and CE to calculate the copy number of survival motor neuron (SMN1 and SMN2) genes for clinical screening of spinal muscular atrophy (SMA). In this study, one universal fluorescent primer was designed and applied for multiplex PCR of SMN1, SMN2 and two internal standards (CYBB and KRIT1). These amplicons were separated by conformation sensitive CE. Mixture of hydroxyethyl cellulose and hydroxypropyl cellulose were used in this CE system. Our method provided the potential to separate two 390‐bp PCR products that differ in a single nucleotide. Differentiation and quantification of SMN1 and SMN2 are essential for clinical screening of SMA patients and carriers. The DNA samples included 22 SMA patients, 45 parents of SMA patients (obligatory carriers) and 217 controls. For evaluating accuracy, those 284 samples were blind‐analyzed by this method and denaturing high pressure liquid chromatography (DHPLC). Eight of the total samples showed different results. Among them, two samples were diagnosed as having only SMN2 gene by DHPLC, however, they contained both SMN1 and SMN2 by our method. They were further confirmed by DNA sequencing. Our method showed good agreement with the DNA sequencing. The multiplex ligation‐dependent probe amplification (MLPA) was used for confirming the other five samples, and showed the same results with our CE method. For only one sample, our CE showed different results with MLPA and DNA sequencing. One out of 284 samples (0.35%) belonged to mismatching. Our method provided a better accurate method and convenient method for clinical genotyping of SMA disease.

Analysis of carbamazepine and its five metabolites in serum by large-volume sample stacking-sweeping capillary electrophoresis.

AbstractThis study establishes a method, using different buffer conductivities and large-volume sample stacking (LVSS)–sweeping capillary electrophoresis, for analysis of carbamazepine (CBZ) and its five metabolites in serum. The capillary (50/60xa0cm) was filled with a high concentration of background electrolyte (150xa0mM phosphate, pHu20093.5, containing 15xa0% methanol), followed by a large volume of samples (10xa0psi, 20xa0s) with low-concentration buffers (5xa0mM phosphate, pHu20093.5, with 5xa0% methanol). When high voltage was applied (−20xa0kV), the sodium dodecyl sulfate (SDS) started to sweep the analytes to an outlet. Meanwhile, the analytes decelerated at the boundary between low- and high-conductivity buffers. Finally, a narrow sample zone was formed. The procedure of sweeping and separation was simultaneously carried out by a sweeping buffer (150xa0mM phosphate, pHu20093.5) with 15xa0% methanol and 50xa0mM SDS added, and the detection was performed by UV at 214xa0nm. The method was validated for linearity (ru2009≧u20090.997), precision, and accuracy. The calibration curves were established for CBZ and its five metabolites between 0.03–25 and 0.03–3xa0μg/mL. The limits of detection (S/Nu2009=u20093) were 0.01xa0μg/mL for each analyte. Compared with simple MEKC (0.5xa0psi, 5xa0s), this system can improve the sensitivity about 300-fold. Finally, this method was successfully applied to five patients, who had taken 200xa0mg CBZ daily, and CBZ levels were found to be from 3.72 to 5.82xa0μg/mL.n FigureChromatogram of resolution of analytes extracted from serum by LVSS-sweeping CE.; peaks: 1. CBZ, 2. CM-3, 3. CM-E, 4. CM-2, 5. CM-10, 6. CM-D, IS: ethyl paraben

Analysis of anabolic androgenic steroids in urine by full-capillary sample injection combined with a sweeping CE stacking method

This study describes an on-line stacking CE approach by sweeping with whole capillary sample filling for analyzing five anabolic androgenic steroids in urine samples. The five anabolic steroids for detection were androstenedione, testosterone, epitestosterone, boldenone, and clostebol. Anabolic androgenic steroids are abused in sport doping because they can promote muscle growth. Therefore, a sensitive detection method is imperatively required for monitoring the urine samples of athletes. In this research, an interesting and reliable stacking capillary electrophoresis method was established for analysis of anabolic steroids in urine. After liquid–liquid extraction by n-hexane, the supernatant was dried and reconstituted with 30xa0mM phosphate buffer (pHxa05.00) and loaded into the capillary by hydrodynamic injection (10xa0psi, 99.9xa0s). The stacking and separation were simultaneously accomplished at −20xa0kV in phosphate buffer (30xa0mM, pHxa05.0) containing 100xa0mM sodium dodecyl sulfate and 40xa0% methanol. During the method validation, calibration curves were linear (ru2009≥u20090.990) over a range of 50–1,000xa0ng/mL for the five analytes. In the evaluation of precision and accuracy for this method, the absolute values of the RSD and the RE in the intra-day (nu2009=u20093) and inter-day (nu2009=u20095) analyses were all less than 6.6xa0%. The limit of detection for the five analytes was 30xa0ng/mL (S/Nu2009=u20095, sampling 99.9xa0s at 10xa0psi). Compared with simple MECK, this stacking method possessed a 108- to 175-fold increase in sensitivity. This simple and sensitive stacking method could be used as a powerful tool for monitoring the illegal use of doping.

Identification of bidirectional gene conversion between SMN1 and SMN2 by simultaneous analysis of SMN dosage and hybrid genes in a Chinese population

Spinal muscular atrophy (SMA) is a neurodegenerative disease characterized by programmed motoneuron death. The survival motor neuron 1 (SMN1) gene is an SMA-determining gene and SMN2 represents an SMA-modifying gene. Here, we applied capillary electrophoresis to quantify the SMN gene dosage in 163 normal individuals, 94 SMA patients and 138 of their parents. We further quantified exons 7 and 8 in SMN1 and SMN2. We found that the SMA patients carried the highest SMN2 copies, which was inversely correlated with disease severity among its three subtypes. Increased SMN1 was significantly associated with decreased SMN2 in the normal group. We also observed that parents of type I SMA patients had significantly fewer SMN2 copies than those of types II and III patients. The hybrid SMN genes were detected in two normal individuals and one patient and her mother. These results imply that increased SMN2 copies in SMA patient group might be derived from SMN1-to-SMN2 conversion, whereas the trend that normal individuals with higher SMN1 copies simultaneously carry fewer SMN2 copies suggested a reverse conversion, SMN2-to-SMN1. Together with the identification of hybrid SMN genes, our data provided additional evidence to support that SMN1 and SMN2 gene loci are interchangeable between population groups.