Novel SPE purification approach using the direct adsorption of vaporised propionic acid in food for rapid HPLC determination.

Solid phase extraction (SPE) is a technique widely used in food analysis for the isolation of analytes. Herein, we proposed a novel application of SPE to extract vaporised propionic acid, a common preservative, from a heated sample solution. A sample was heated under acidified conditions and the resulting steam was directly passed through an SPE column to extract the propionic acid, followed by elution and HPLC analysis. Here, the extraction on the SPE column ensures direct capture of propionic acid. The results demonstrated excellent linearity (R2 greater than 0.999) and recoveries of 89.9%-97.6% with intra- and inter-day precisions lower than 3.9%. To the best of our knowledge, no study has investigated the applicability of SPE to an analyte vaporised in the headspace of food products. The proposed method is promising in its application to various volatile compounds and in the routine analysis of propionic acid in food.

[Internal Quality Control of Residual Pesticides Analysis Using Multiple Stable Isotope Labeled Compounds].

Internal quality control (IQC) is essential to ensure the reliability of the results of chemical analysis. In this study, we propose a novel method of IQC for multiresidue analysis of pesticides. A total of seven stable isotope labeled compounds (SILC) were added to analytical samples and were used to monitor and evaluate the quality of analytical results. In contrast to conventional IQC method in which only a limited number of control materials were analyzed to ensure the reliability of the results for an entire batch, the developed method can monitor the analytical quality of all the samples in the batch. It was shown that the developed method could achieve better performance than that of conventional method. Therefore, the developed method is considered to be promising for practical applications.(Received January 27, 2022; Accepted July 4, 2022).

Combined use of a modifier gas generator, analyte protectants and multiple internal standards for effective and robust compensation of matrix effects in gas chromatographic analysis of pesticides.

In the analysis of pesticides performed with gas chromatography, the quantitative performance of measurements can be severely compromised by phenomena known as matrix effects. In seeking a solution to the problem of matrix effects, the application of a modifier gas generator (MGG) was investigated in this study, together with analyte protectants and multiple internal standards. Ethylene glycol (EG) was used as modifier gas and matrix effects in GCMS analysis were then evaluated by using the extracts of various food commodities. MGG was used in combination with other known methods of matrix effect compensation and its performance in reducing matrix effects tested. We have found that by combining MGG with conventional analyte protectants, matrix effects were substantially reduced for most of pesticides. Use of EG was especially effective for organophosphate pesticides and those with amino groups. Using this approach, the shortcomings of conventional analyte protectants were remedied. Although neither EG nor analyte protectants could sufficiently reduce the matrix effects for certain classes of pesticides, this limitation could be overcome with the use of multiple internal standards (IS) in the analysis. Finally, it was shown that the method we developed could achieve better analytical performance than the matrix-matched calibration method. Our method was robust with respect to the variation of food matrix components, so its application to real-world analyses would be practical and promising.

Simultaneous determination of nine preservatives in food by liquid chromatography with the aid of coagulant in the clean-up process.

A simple HPLC method was developed for the simultaneous analysis of nine preservatives in food including benzoic acid, sorbic acid, dehydroacetic acid, methyl paraben, ethyl paraben, isopropyl paraben, propyl paraben, isobutyl paraben and butyl paraben. Samples were extracted with 60 v/v% methanol containing poly-aluminium chloride (PAC) and sodium hydroxide prior to analysis. PAC, which is normally used as a coagulant, was successfully applied to remove interfering substances from the samples. The method showed good linearity with coefficients of determination higher than 0.999 over the range of 0.2-5 µg ml-1 for all target preservatives. LOQs of the method were in the range of 0.002-0.008 g kg-1. Method performance was evaluated in a variety of foods demonstrated to have quantitative recoveries of 81.7-102.5% with satisfactory intra-day precision of < 3.7% and inter-day precision of < 6.5%. The method also demonstrated applicability to real foods containing preservatives.

[Development of a New Method for Determination of Sodium Saccharin and Acesulfame Potassium with the Aid of Coagulant].

An HPLC method for determination of sodium saccharin and acesulfame potassium was newly developed, employing coagulant pretreatment to remove particles dispersed in the sample extract. The method showed recovery of 96-101% for both analytes with a repeatability of less than 1% and a reproducibility of less than 2%. The limit of quantification for sodium saccharin was 0.025 g/kg and that for acesulfame potassium was 0.025 g/kg. Only about 20 min was required for preparation of the test solution, whereas the dialysis method takes much longer.

[Application of Coagulant in the Analysis of Lycorine and Galanthamine in Processed Foods].

Analytical method by HPLC and LC-MS/MS for determining lycorine and galanthamine in processed food was newly developed. In this method, coagulant which has never been used in food analysis was applied on cleanup process. With coagulant approach, removal of interfering substances on determination for analytes was easily achieved. The method using HPLC showed recovery of 95.4-102.9% on both analytes with repeatability of less than 2.9% and reproducibility of less than 2.9%. The method using LC-MS/MS showed recovery of 97.4-107.6% with repeatability of less than 5.7% and reproducibility of less than 5.7%. On HPLC method, limit of quantification for lycorine was 0.004 g/kg and that of galanthamine was 0.006 g/kg. On LC-MS/MS method, limit of quantification for lycorine was 0.0008 g/kg and that of galanthamine was 0.0005 g/kg.

Purification and characterization of cellobiose dehydrogenase from white-rot basidiomycete Trametes hirsuta.

In order to save energy during the pulp making process, we tried to use white-rot basidiomycete, Trametes hirsuta, which degrades lignin efficiently. But a decrease in paper strength caused by cellulolytic activity ruled this out for practical application. Since the cellulolytic activity of the fungus must be decreased, we purified and characterized a cellobiose dehydrogenase (CDH) that was reported to damage pulp fiber. The CDH in the culture filtrate of C. hirsutus was purified by freeze-thawing and chromatographic methods. The pI of the enzyme was 4.2 and its molecular weight was 92 kDa. The optimal temperature and pH of the enzyme were 60-70 degrees C and 5.0 respectively. Since the purified CDH decreased the viscosity of pulp in the presence of Fe(III) and cellobiose, it was shown that the suppression of CDH should be an effective way to reduce cellulose damage.

Intron-dependent accumulation of mRNA in Coriolus hirsutus of lignin peroxidase gene the product of which is involved in conversion/degradation of polychlorinated aromatic hydrocarbons.

The homobasidiomycete Coriolus hirsutus coding sequences of a lignin peroxidase (LiP) gene (lip, containing six (I-VI) introns), a lip cDNA (lipc), and three lipc derivatives containing one (I), three (I-III), or five (I-V) introns were inserted into chromosome-integrating expression vector. These recombinant plasmids were introduced into C. hirustus monokaryotic strain. The transformant carrying the promoter-lipc-terminator cassette did not contain enough mRNA molecules to be detectable by Northern-blot analysis. On the other hand, all the transformants carrying cassettes of genomic lip and intron(s)-containing lipc sequences contained sufficient amounts of mRNAs to be easily detected by Northern-blot analysis. LiP activities in the culture supernatants of these transformants were found to be about five times as high as those of transformants carrying the lipc cassette (or no cassette). The culture supernatants of the transformants with high LiP activity showed remarkably high conversion activity toward pentachlorophenol (PCP) and degradation activity toward 2,7-dichlorodibenzo-p-dioxin (2,7-DCDD). These results indicate that at least one intron (intron I) is required for accumulation of lip mRNA and its subsequent translational expression in C. hirsutus.

Rat cytochrome P450-mediated transformation of dichlorodibenzo-p-dioxins by recombinant white-rot basidiomycete Coriolus hirsutus.

Rat cytochrome P450, CYP1A1, has been reported to play an important role in the metabolism of mono-trichlorodibenzo-p-dioxins (M-TriCDDs). To breed lignin (and M-TetraCDDs)-degrading basidiomycete Coriolus hirsutus strains producing rat CYP1A1, an expression cassette [C. hirsutus gpd promoter-C. hirsutus gpd 5' portion (224-bp of 1st exon-8th base of 4th exon)-rat cyp1a1 cDNA-Lentinula edodes priA terminator] was constructed and inserted into pUCR1 carrying the C. hirsutus arg1 gene. The resulting recombinant plasmid, MIp5-(cyp1a1 + arg1) was introduced into protoplasts of C. hirsutus monokaryotic strain OJ1078 (Arg(-), Leu(-)), obtaining three good Arg(+) transformants. These transformants [ChTF5-2(CYP1A1), ChTF5-4(CYP1A1), and ChTF5-6(CYP1A1)] were estimated to carry nine, six, and seven copies of the expression cassette on their chromosomes, respectively. Immunoblot analysis revealed that the three transformants produce similar amounts of rat CYP1A1 enzyme. ChTF5-2(CYP1A1), ChTF5-4(CYP1A1), ChTF5-6(CYP1A1) and recipient OJ1078 were cultivated in a liquid medium containing 2,7/2,8(at a ratio of 1:1)-dichlorodibenzo-p-dioxins (2,7/2,8-DCDDs) and the amount of intra- and extracellular 2,7/2,8-DCDDs remaining was measured. The results showed that all three transformants efficiently transform 2,7/2,8-DCDDs through the action of the recombinant rat CYP1A1 enzyme.

Transformation of the white-rot basidiomycete Coriolus hirsutus using the ornithine carbamoyltransferase gene.

An efficient transformation system for the basidiomycete Coriolus hirsutus was developed. A double-auxotrophic mutant of C. hirsutus, deficient both in ornithine carbamoyltransferase (OCTase) and 3-isopropylmalate dehydrogenase (3-IPM dehydrogenase), was transformed to Arg+ with each allelic type of the C. hirsutus genomic OCTase gene (arg1) newly cloned. The transformation frequency of 10(3)-10(4) transformants per mug DNA per 10(6)-10(7) oidial protoplasts was reached. Southern blots showed that the transforming DNA was integrated into chromosomal DNA with multi-copies. The Arg+ phenotype of the transformants was stably inherited through mitosis.