Evaluation of fresh, frozen, and lyophilized fecal samples by SPME and derivatization methods using GC×GC-TOFMS.
Author(s): Nam SL, Tarazona Carrillo K, de la Mata AP, de Bruin OM, Doukhanine E, Harynuk J
Publication: Metabolomics, 2022, Vol. 18, Page 25
PubMed ID: 35426515 PubMed Review Paper? No
Purpose of Paper
This paper compared two-dimensional gas chromatography coupled to time-of-flight mass spectrometry (GC×GC-TOFMS) metabolite profiles among frozen, lyophilized, and fresh aliquots of a single fecal specimen using two different processing methods.
Conclusion of Paper
Solid phase microextraction (SPME) preparation of multiple aliquots of a single fecal specimens resulted in more prominent differences than trimethylsilylation (TMS) derivatization. Using SPME, the average number of peaks detected, total peak area (TPA) and total useful peak area (TUPA) were all highest in the lyophilized aliquots and lowest in the fresh aliquots. The average number of peaks observed was slightly lower in the fresh aliquots compared to lyophilized and frozen aliquots when specimens were processed using TMS derivatization. Relative standard deviation was lowest for frozen aliquots and highest for fresh aliquots using either processing method. The prominent compound classes in the SPME aliquots were SCFAs (C2-C5), fatty acids (C8-C14), ketones (C3-C16), aldehydes (C4-C15), alcohols (C4-C13), sulfides, and terpenes while the most prominent compounds using the TMS derivatization protocol were amino acids, fatty acids (C5- C18), carbohydrates, bile acids, tocopherols, and sterols. Using the SPME protocol, most metabolites were found at higher levels in lyophilized aliquots while sulfide levels were comparable among frozen, lyophilized and fresh aliquots; however, the magnitude of differences between differentially preserved aliquots was peak dependent. Differences between preservation methods were generally small (<3 fold) in specimens processed by TMS derivatization but five N-acetyl glucosamine-4TMS peaks were 5-10 fold higher in the lyophilized and frozen aliquots compared to fresh aliquots.
Studies
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Study Purpose
This study compared the GC×GC-TOFMS metabolite profiles among frozen, lyophilized and fresh aliquots of a single fecal specimen using two different processing methods (SPME and TMS derivatization). A single fecal specimen was obtained from a healthy volunteer and immediately homogenized by stirring. The specimen was then divided into three aliquot which were analyzed within 1 h, stored at -80°C for 82 days or stored at -80°C for 8 h before being lyophilized and stored at -80°C. Lyophilized specimens were reconstituted with water before solid-phase microextraction (SPME). For each aliquot SPME was performed sequentially on four replicates using an automated SPME system. Aliquots of feces (6 per condition) were mixed with 80% methanol, centrifuged, dried under nitrogen at 37°C, and stored at -80°C until derivation. During TMS derivatization, specimens were mixed with anhydrous toluene, dried under nitrogen at 50°C, mixed with methoxyamine hydrochloride, incubated at 80°C for 30 min. SPME and TMS derivation specimens were analyzed by GC×GC-TOFMS within 24 h. Data processing was performed using ChromaTOF using separate pathways for SPME and TMS derivatization.
Summary of Findings:
The storage conditions evaluated for fecal specimens resulted in more prominent differences in spectra when the SPME preparation method was used compared to TMS derivatization. Using the SPME preparation method, the average number of peaks detected, TPA and TUPA were all highest in the lyophilized fecal aliquots (1282, 1.79 x 107, and 1.22 x 107, respectively), followed by the frozen aliquots (803, 9.13 x 106, and 5.10 x 106, respectively) and then the fresh aliquot (588, 4.88 x 106, and 3.11 x 106, respectively). The authors attribute the higher number of peaks in lyophilized and frozen aliquots to the release of metabolites during the freezing/thawing of the specimen. In contrast to SPME, there were no obvious differences in chromatographs of lyophilized, frozen and fresh aliquots processed by TMS derivatization, but the average number of peaks observed was still slightly lower in fresh aliquots (1238) compared to lyophilized (1357) and frozen (1307) aliquots. The relative standard deviation was lowest for frozen aliquots and highest for fresh aliquots using either processing methods. The prominent compound classes in the SPME specimens were SCFAs (C2-C5), fatty acids (C8-C14), ketones (C3-C16), aldehydes (C4-C15), alcohols (C4-C13), sulfides, and terpenes while the most prominent compounds using the TMS derivatization protocol were amino acids, fatty acids (C5- C18), carbohydrates, bile acids, tocopherols, and sterols. Using the SPME protocol, most metabolites were found at higher levels in lyophilized aliquots although sulfide levels were comparable among frozen, lyophilized and fresh aliquots; however, the magnitude of the differences between differentially preserved aliquots was peak dependent. While differences between preservation methods were generally small (<3 fold) in TMS derivatization specimens, five N-acetyl glucosamine-4TMS peaks were 5-10 fold higher in lyophilized and frozen aliquots than fresh aliquots.
Biospecimens
Preservative Types
- Frozen
- Other Preservative
- None (Fresh)
Diagnoses:
- Normal
Platform:
Analyte Technology Platform Steroid GC-TOF-MS Small molecule GC-TOF-MS Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Preservation Type of fixation/preservation Lyophilized
Frozen
None (fresh)
Analyte Extraction and Purification Analyte isolation method SPME
TMS derivatization