Determination of Tryptophan Metabolites in Serum and Cerebrospinal Fluid Samples Using Microextraction by Packed Sorbent, Silylation and GC–MS Detection
Abstract
:1. Introduction
2. Results and Discussion
2.1. Mass Spectrometric Characteristics of the Silyl Derivatives of Indolic Acids
2.2. Retrospective Analysis of Indolic Acids in Biological Samples of Critically Ill Patients
2.3. Analytical Characteristics of MEPS for Indolic Acid Extraction
2.4. Application of MEPS for Indolic Acid Extraction from Biological Samples of Critically Ill Patients
- CSF samples: #1–(0.42 ± 0.08) µM; #2–(0.6 ± 0.1) µM; #3–(0.43 ± 0.03) µM.
- Serum samples: #A–(0.47 ± 0.09) µM; #B–(0.43 ± 0.06) µM; #C–(0.6 ± 0.1) µM.
2.5. MEPS for Reducing the Matrix Effect
3. Materials and Methods
3.1. Chemicals and Reagents
3.2. Analysis of Model and Biological Samples
3.3. MEPS Procedure
3.4. Method Validation
3.4.1. Recovery
3.4.2. Calibration Curve
3.4.3. LOD and LOQ
3.4.4. Accuracy and Precision
3.4.5. Selectivity
3.4.6. Stability
3.4.7. Carryover Effects
3.5. Statistical Analysis
4. Conclusions
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
BSTFA | N,O-bis(trimethylsilyl)trifluoroacetamide |
CNS | central nervous system |
CSF | cerebrospinal fluid |
FDA | Food and Drug Administration |
GC–MS | gas chromatography–mass spectrometry |
5HIAA | 5-hydroxyindole-3-acetic acid |
HMDB | Human Metabolome Database |
3IAA | indole-3-acetic acid |
3ICA | indole-3-carboxylic acid |
3ILA | indole-3-lactic acid |
3IPA | indole-3-propionic acid |
LLE | liquid-liquid extraction |
LOD | limit of detection |
LOQ | limit of quantitation |
MEPS | microextraction by packed sorbent |
MTBSTFA | N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide |
NIST | National Institute of Standards and Technology |
RE | relative error |
RSD | relative standard deviation |
Rt | retention time |
TBDMS | tert-butyldimethylsilyl |
TMS | trimethylsilyl |
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Sample Availability: Samples of the compounds are not available from the authors. |
Indolic Acid, Derivative, Structure | Rt, min | M, Da | m/z | Possible Structure of Characteristic Ions |
---|---|---|---|---|
Trimethylsilyl (TMS) Derivatives | ||||
Indole-3-acetic acid (3IAA), mono- (CH3)3SiOOCCH2-C8H6N | 18.26 | 247 | 130 247 | [M-COOSi(CH3)3]+ M+ |
3IAA, di- (CH3)3SiOOCCH2-C8H5NSi(CH3)3 | 18.60 | 319 | 202 319 | [M-COOSi(CH3)3]+ M+ |
Indole-3-carboxylic acid (3ICA), mono-1 (CH3)3SiOOC-C8H6N | 19.12 | 233 | 218 233 | [M-CH3]+ M+ |
3ICA, di- (CH3)3SiOOC-C8H5NSi(CH3)3 | 19.21 | 305 | 290 305 | [M-CH3]+ M+ |
Indole-3-propionic acid (3IPA), mono-1 (CH3)3SiOOC(CH2)2-C8H6N | 19.44 | 261 | 130 261 | [M-CH2COOSi(CH3)3]+ M+ |
3IPA, di- (CH3)3SiOOC(CH2)2-C8H5NSi(CH3)3 | 19.77 | 333 | 202 333 | [M-CH2COOSi(CH3)3]+ M+ |
Indole-3-lactic acid (3ILA), di-1 NC8H6-CH2CH(OSi(CH3)3)COOSi(CH3)3 | 20.35 | 349 | 130 | [M-CH(OSi(CH3)3)-COOSi(CH3)3]+ |
3ILA, tri- (CH3)3Si-NC8H5-CH2CH(OSi(CH3)3)COOSi(CH3)3 | 20.49 | 421 | 202 421 | [M-CH(OSi(CH3)3)-COOSi(CH3)3]+ M+ |
5-Hydroxyindole-3-acetic acid (5HIAA), tri- (CH3)3SiOOCCH2-C8H4NSi(CH3)3-OSi(CH3)3 | 20.88 | 407 | 290 407 | [M-OSi(CH3)3; -CH3]+ M+ |
5HIAA, di- (CH3)3SiOOCCH2-C8H5N-OSi(CH3)3 | 20.99 | 335 | 218 335 | [M-COOSi(CH3)3]+ M+ |
Tert-butyldimethylsilyl (TBDMS) Derivatives | ||||
3IAA, mono- (CH3)3C-(CH3)2SiOOCCH2-C8H6N | 20.54 | 289 | 130 232 289 | [M-COOSi(CH3)3-C(CH3)3]+ [M-C(CH3)3]+ M+ |
3ICA, mono-1 (CH3)3C-(CH3)2SiOOC-C8H6N | 21.52 | 275 | 218 | [M-C(CH3)3]+ |
3IPA, mono-1 (CH3)3C-(CH3)2SiOOC(CH2)2-C8H6N | 21.63 | 303 | 130 246 303 | [M-CH2COOSi(CH3)3-C(CH3)3]+ [M-C(CH3)3]+ M+ |
3ILA, di- NC8H6-CH2CH(OSi(CH3)2- C(CH3)3)COOSi(CH3)2-C(CH3)3 | 23.83 | 433 | 130 376 | [M-CH(OSi(CH3)3-C(CH3)3)-COOSi(CH3)3-C(CH3)3]+ [M-C(CH3)3]+ |
5HIAA, di- (CH3)3C-(CH3)2SiO-NC8H5-CH2COOSi(CH3)2-C(CH3)3 | 24.38 | 419 | 362 419 | [M-C(CH3)3]+M+ |
Group of Patients | Number of Samples | Presence in Samples | mono-TMS (1) | di-TMS (2) | 1 + 2 | A1 ˃ A2 | A1 ˂ A2 |
---|---|---|---|---|---|---|---|
1 | 38 | 35 | 21 | 27 | 13 | 8 | 5 |
2 | 24 | 23 | 8 | 23 | 7 | 4 | 3 |
3 | 64 | 59 | 58 | 14 | 13 | 11 | 2 |
4 | 55 | 55 | 51 | 53 | 49 | 31 | 18 |
5 | 107 | 91 | 56 | 70 | 35 | 25 | 10 |
Σ | 288 | 263 | 194 | 187 | 118 | 79 | 38 |
Indolic Acid Derivative | Model Solution | Pooled CSF | Blood Serum | |||||
---|---|---|---|---|---|---|---|---|
BSTFA | MTBSTFA | 100 a | 500 b | 1500 c | 100 a | 500 b | 2000 d | |
Indole-3-acetic acid (3IAA), mono- | 80 ± 20 | 80 ± 10 | 80 ± 20 | 80 ± 20 | 80 ± 10 | 60 ± 20 | 60 ± 10 | 60 ± 20 |
Indole-3-carboxylic acid (3ICA), mono- | 70 ± 10 | 70 ± 20 | 60 ± 20 | 70 ± 20 | 60 ± 10 | 60 ± 10 | 70 ± 20 | 60 ± 10 |
Indole-3-propionic acid (3IPA), mono- | 62 ± 6 | 40 ± 10 | 70 ± 20 | 70 ± 10 | 70 ± 10 | 48 ± 9 | 50 ± 10 | 50 ± 10 |
Indole-3-lactic acid (3ILA), di- | 80 ± 20 | 70 ± 20 | 80 ± 20 | 70 ± 20 | 80 ± 20 | 50 ± 10 | 50 ± 10 | 60 ± 10 |
5-Hydroxyindole-3-acetic acid (5HIAA), di- | 52 ± 8 | 40 ± 10 | 40 ± 10 | 50 ± 10 | 50 ± 10 | 40 ± 10 | 38 ± 6 | 40 ± 10 |
Indolic Acid | Matrix | Dynamic Concentration Range, µM | R2 | LOD, µM | LOQ, µM | 1RSD, % | 1RE, % |
---|---|---|---|---|---|---|---|
Indole-3-acetic acid (3IAA) | Model solution | 0.4–9 | 0.9982 | 0.1 | 0.4 | 15 | −9 |
Pooled CSF | 0.4–7 | 0.9951 | 0.2 | 0.4 | 16 | −11 | |
Blood serum | 0.4–9 | 0.9945 | 0.2 | 0.4 | 19 | 15 | |
Indole-3-carboxylic acid (3ICA) | Model solution | 0.5–10 | 0.9973 | 0.1 | 0.5 | 7 | 10 |
Pooled CSF | 0.5–7 | 0.9998 | 0.3 | 0.5 | 10 | 20 | |
Blood serum | 0.5–10 | 0.9987 | 0.4 | 0.5 | 20 | −17 | |
Indole-3-propionic acid (3IPA) | Model solution | 0.4–8 | 0.9989 | 0.1 | 0.4 | 6 | 13 |
Pooled CSF | 0.4–6 | 0.9949 | 0.4 | 0.4 | 18 | 16 | |
Blood serum | 0.4–8 | 0.9942 | 0.4 | 0.4 | 16 | 12 | |
Indole-3-lactic acid (3ILA) | Model solution | 0.4–8 | 0.9960 | 0.2 | 0.4 | 17 | 19 |
Pooled CSF | 0.4–6 | 0.9963 | 0.2 | 0.4 | 18 | −20 | |
Blood serum | 0.4–8 | 0.9967 | 0.3 | 0.4 | 18 | 13 | |
5-Hydroxyindole-3-acetic acid (5HIAA) | Model solution | 0.4–8 | 0.9981 | 0.1 | 0.4 | 7 | −11 |
Pooled CSF | 0.4–6 | 0.9991 | 0.3 | 0.4 | 19 | 19 | |
Blood serum | 0.4–8 | 0.9991 | 0.4 | 0.4 | 20 | 17 |
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Pautova, A.; Khesina, Z.; Getsina, M.; Sobolev, P.; Revelsky, A.; Beloborodova, N. Determination of Tryptophan Metabolites in Serum and Cerebrospinal Fluid Samples Using Microextraction by Packed Sorbent, Silylation and GC–MS Detection. Molecules 2020, 25, 3258. https://doi.org/10.3390/molecules25143258
Pautova A, Khesina Z, Getsina M, Sobolev P, Revelsky A, Beloborodova N. Determination of Tryptophan Metabolites in Serum and Cerebrospinal Fluid Samples Using Microextraction by Packed Sorbent, Silylation and GC–MS Detection. Molecules. 2020; 25(14):3258. https://doi.org/10.3390/molecules25143258
Chicago/Turabian StylePautova, Alisa, Zoya Khesina, Maria Getsina, Pavel Sobolev, Alexander Revelsky, and Natalia Beloborodova. 2020. "Determination of Tryptophan Metabolites in Serum and Cerebrospinal Fluid Samples Using Microextraction by Packed Sorbent, Silylation and GC–MS Detection" Molecules 25, no. 14: 3258. https://doi.org/10.3390/molecules25143258