INVESTIGATIONS OF URINARY METABOLIC PROFILING USING GAS CHROMATOGRAPHY/MASS SPECTROMETRY: A PRETREATMENT METHOD INVESTIGATRION

As one of the most widely adopted techniques in metabonomics, gas chromatography/mass spectrometry (GC/MS) combines the advantages of GC and MS, which includes high-efficiency separation and resolution, as well as informative mass spectral of reference compounds for metabolites identification, providing the basis for gaining multitudinous metabolites. In the meantime, attentions have to be paid to the stability and reproducibility of GC/MS based metabonomics as well, which has a great impact on the accuracy and validation of metabonomic investigations. Furthermore, urine is frequently applied in metabonomic research. Accordingly, stability and reproducibility of urinary metabolic profiling were studied using gas chromatography/time of flight mass spectrometry (GC/TOFMS) in this work. It was demonstrated that the activity of urease dissolved in ultrapure water could be maintained for at least 15 days, however, urease prepared in phosphate buffer (pH=7.0) remained active only for 5 days under the same condition at -20℃. Ethylmalonic acid, glycerol, 1,2,3-butantriol, L-isoleucine, L-threonine, glycine, succinic acid, which were overlaid by urea in the urine samples without urease treatment, could be effectively isolated and identified after urease treatment. Nevertheless, the amount of detected metabolites and the contents of majority of the metabolites all decreased with urea degradation. Additionally, metabolites in urine with urea treatment for 15 min are more stable compared to 30 min and 60 min according to the results of relative standard deviation (RSD) distribution of ion peak areas. On the other hand, it is clear from Fig.1. that metabolites in two-step derivatized samples are more stable than those in one-step samples. The average RSD of peak areas of ions in two-step derivatized samples is 13.10±14.62, significantly lower than that in one-step samples. And the number of ion peaks (RSD of peak areas<20%) from two-step derivatization samples is 1509, obviously larger than that from one-step samples. Besides, the volume ratio of methanol to urinary incubation solutions was studied. It was found that repeatability and stability became higher with the increase of the ratio of methanol to urinary solutions. In conclusion, high repeatability and stability of urinary metabolic profiling based on GC/MS techniques could be obtained by employing the following preprocessing conditions. Urease is prepared in ultrapure water; the time of urease treatment is 15 min; ratio of methanol to urinary incubation solutions is 4:1; and two-step derivatization is employed.As one of the most widely adopted techniques in metabonomics, gas chromatography/mass spectrometry (GC/MS) combines the advantages of GC and MS, which includes high-efficiency separation and resolution, as well as informative mass spectral of reference compounds for metabolites identification, providing the basis for gaining multitudinous metabolites. In the meantime, attentions have to be paid to the stability and reproducibility of GC/MS based metabonomics as well, which has a great impact on the accuracy and validation of metabonomic investigations. Furthermore, urine is frequently applied in metabonomic research. Accordingly, stability and reproducibility of urinary metabolic profiling were studied using gas chromatography/time of flight mass spectrometry (GC/TOFMS) in this work. It was demonstrated that the activity of urease dissolved in ultrapure water could be maintained for at least 15 days, however, urease prepared in phosphate buffer (pH=7.0) remained active only for 5 days under the same condition at -20℃. Ethylmalonic acid, glycerol, 1,2,3-butantriol, L-isoleucine, L-threonine, glycine, succinic acid, which were overlaid by urea in the urine samples without urease treatment, could be effectively isolated and identified after urease treatment. Nevertheless, the amount of detected metabolites and the contents of majority of the metabolites all decreased with urea degradation. Additionally, metabolites in urine with urea treatment for 15 min are more stable compared to 30 min and 60 min according to the results of relative standard deviation (RSD) distribution of ion peak areas. On the other hand, it is clear from Fig.1. that metabolites in two-step derivatized samples are more stable than those in one-step samples. The average RSD of peak areas of ions in two-step derivatized samples is 13.10±14.62, significantly lower than that in one-step samples. And the number of ion peaks (RSD of peak areas