A ninety-six well plate as headspaces with moist starch indicator paper as a cover for the determination of ascorbic acid by iodate oxidation and formation of volatile iodine.

This work presents the use of a 96-well plate as headspaces for the determination of ascorbic acid in samples loaded in the 96-well plate. Ascorbic acid in the sample is oxidized to iodide by the addition of excess acidic iodate solution into the well. The iodide is further oxidized by the remaining iodate to molecular iodine. A single sheet of moist starch indicator paper is immediately placed over the 96-well plate after the addition of the iodate with the moisture forming a gas seal. The iodine gas in each well diffuses through the headspace to react with the starch paper producing circular areas of a colored starch-iodine complex. After 15 min the indicator paper is scanned, and the digital images of the complex are analyzed by using ImageJ software to obtain blue intensity values. The precision of the intensity values from 12 wells containing 20 µL of 2.84 mM standard ascorbic acid is <2% relative standard deviation. Optimal conditions for detection were investigated, including the starch concentration, the acidic iodate reagent, and the measurement time. The linear calibration range of ascorbic acid is 0.284-2.84 mM, based on the plot of concentration vs. -log(reflectance). The coefficient of determination (r2) is >0.998. Samples of fruit juice and dietary supplements were analyzed for their ascorbic acid contents. The results obtained from the headspace reflectance method are not statistically different from values obtained from the titration method using paired t-tests (α = 0.05).

Development of a solid-phase extraction method with simple MEKC-UV analysis for simultaneous detection of indole metabolites in human urine after administration of indole dietary supplement.

This work presents the development of a solid phase extraction method with simple MEKC-UV analysis for the simultaneous determination of indole-3-carbinol (I3C) and its metabolites (3, 3'-diindolylmethane (DIM), indole-3-carboxaldehyde (I3CAL), indole-3-acetonitrile (I3A)) in human urine after oral administration of an indole dietary supplement. Solid phase extraction (SPE) method was applied for the first time for simultaneous analysis of these indole metabolites. The MEKC separation method was developed in a previous work. Three commercial SPE cartridges, each with different sorbent materials, were investigated: Sep-Pak® C18, Oasis® HLB and Oasis® WCX. The Sep-Pak® C18 material provided the highest extraction recovery of 88-113% (n = 9), for the four target indole metabolites (I3C, DIM, I3CAL and I3A). The optimal washing and elution solutions were 40% methanol/water (v/v) and 100% methanol, respectively, and optimal elution volume was 2.0mL. The specificity of the proposed SPE method was evaluated with negative control urine samples (n = 10) from healthy volunteers who had not taken the dietary supplement or vegetables known to contain indole compounds. Linear calibration curves were in the range of 0.2-25µgmL-1 (r2 > 0.998) using diphenylamine (DPA) as the internal standard. Intra-day and inter-day precisions were 3.5-12.3%RSD and 2.7-14.1%RSD, respectively. Limits of detection and quantification were 0.05-0.10µgmL-1 and 0.10-0.50µgmL-1, respectively. The four target indole compounds were separated within only 5min by MEKC-UV analysis. Urine from 5 subjects who had taken a dietary supplement containing I3C and DIM were found to contain only the DIM metabolite at concentrations ranging from 0.10 to 0.35µgmL-1. Accuracy of the proposed method based on the percentage recovery of spiked urine samples were 70-108%, 82-116%, 82-132% and 80-100% for I3C, I3CAL, I3A and DIM, respectively. The Sep-Pak®C18 cartridge was highly effective in extraction and sample cleanup for the downstream simultaneous detection of urinary indole metabolites by MEKC-UV method.

Development of a sequential injection-liquid microextraction procedure with GC-FID for analysis of short-chain fatty acids in palm oil mill effluent.

Short-chain fatty acids, such as acetic, propionic, butyric, iso-valeric and valeric acids, play an important role in methanogenesis activity for biogas production processes. Thus, simple and rapid procedures for monitoring the levels of short-chain fatty acids are requisite for sustaining biogas production. This work presents the development of a sequential injection-liquid microextraction (SI-LME) procedure with GC-FID analysis for determination of short-chain fatty acids. GC-FID was employed for detection of the short-chain fatty acids. Calibration curves were linear with good coefficients of determination (r2>0.999), using methacrylic acid as the internal standard. Limits of quantification (LOQ) were in the range of 0.03-0.19mM. The SI-LME procedure employed tert-butyl methyl ether (TBME) as the extracting solvent. Various SI-LME conditions were investigated and optimized to obtain the highest recovery of extraction. With these optimized conditions, an extraction recovery of the five key short-chain fatty acids of 67-90% was obtained, with less than 2% RSD (n=3). The final SI-LME procedure employed two fluidic zones of TBME with a single aqueous fluidic zone of sample sandwiched between the TBME zones, with 5 cycles of flow reversal at a flow rate of 5µL/s for the extraction process. Intra- and inter-day precision values were 0.5-4.0% RSD and 3.3-4.8% RSD, respectively. Accuracy based on percentage of sample recovery were in the range of 69-96, 102-107, and 82-101% (n=4) for acetic, propionic and butyric acids, respectively. The proposed method was applied for the measurement of short-chain fatty acids in palm oil mill effluents used in biogas production in a factory performing palm oil extraction process. The SI-LME method provides improved extraction performance with high precision, and is both simple and rapid with its economical extraction technique. The SI-LME procedure with GC-FID has strong potential for use as a quality control process for monitoring short-chain fatty acid levels in biogas production.