Introduction:
Inborn errors of metabolism (IEM) cause significant morbidity and mortality when left untreated. Many IEMs have pathognomonic urine organic acid (UOA) levels, usually qualitatively analyzed via the current gold standard, GC-EI-MS (Gas Chromatography-Electron Impact-Mass Spectroscopy). The Agilent 7890 GC in tandem with the Waters’ Xevo TQ-S MS contains an easily interchangeable LC-ESI (liquid chromatography-electrospray ionization) and GC-APCI (Atmospheric Pressure Chemical Ionization) instrument set-up that maintains accuracy and sensitivity in both LC and GC modes.
Objectives: 
Utilizing this novel GC-APCI instrument, this project aims to develop and validate a new UOA method for clinical use with the goal of transitioning to a quantitative method using multi-reaction monitoring (MRM) mode.
Methods:
Chemical standards and patient urine samples were initially spiked with tropic acid, the internal standard. UOAs were then extracted via a liquid-liquid ether extraction and reacted with BSTFA to form trimethylsilyl derivatives. Initial instrument suitability and method setup was assessed and compared on both the current GC-EI-MS and the new GC-APCI-MS. Source moisture levels were modified to explore the wet proton transfer mechanism, [M+H]+, and the dry charge transfer mechanism, [M*]+. Following machine optimization, the elution times and ion mass spectra profiles of metabolites of interest were determined in preparation for MRM mode.
Results:
Initial patient sample experiments achieved successful separation of key isobaric UOAs. During wet and dry mode exploration, the former had greater peak areas and signal-to-noise ratios. 61 organic acids of clinical interest were evaluated in patient and laboratory quality control samples. These metabolites were detected to high sensitivity following transition to MRM mode and methyl-malonic acid (MMA) was chosen for initial quantitative analysis. With a R squared of 0.9776 for MMA, this instrument shows strong linearity in the clinical ranges of 10-10000 uM.
Conclusion:
The Waters’ Xevo TQ-S micro with Agilent 7890 GC demonstrated promising GC-resolution separation of UOA metabolites coupled to APCI-MS detection. Prior to clinical use, APCI library expansion and further MRM-based quantitative assays need to be done. With clear avenues for future work, the APCI technique offers great benefits for the field of metabolics with LC and GC analysis on a single instrument while improving newborn patient outcome.

**Introduction:**
Inborn errors of metabolism (IEM) cause significant morbidity and mortality when left untreated. Many IEMs have pathognomonic urine organic acid (UOA) levels, usually qualitatively analyzed via the current gold standard, GC-EI-MS (Gas Chromatography-Electron Impact-Mass Spectroscopy). The Agilent 7890 GC in tandem with the Waters’ Xevo TQ-S MS contains an easily interchangeable LC-ESI (liquid chromatography-electrospray ionization) and GC-APCI (Atmospheric Pressure Chemical Ionization) instrument set-up that maintains accuracy and sensitivity in both LC and GC modes.
**Objectives:** 
Utilizing this novel GC-APCI instrument, this project aims to develop and validate a new UOA method for clinical use with the goal of transitioning to a quantitative method using multi-reaction monitoring (MRM) mode.
**Methods:**
Chemical standards and patient urine samples were initially spiked with tropic acid, the internal standard. UOAs were then extracted via a liquid-liquid ether extraction and reacted with BSTFA to form trimethylsilyl derivatives. Initial instrument suitability and method setup was assessed and compared on both the current GC-EI-MS and the new GC-APCI-MS. Source moisture levels were modified to explore the wet proton transfer mechanism, [M+H]^+^, and the dry charge transfer mechanism, [M*]^+^. Following machine optimization, the elution times and ion mass spectra profiles of metabolites of interest were determined in preparation for MRM mode.
**Results:**
Initial patient sample experiments achieved successful separation of key isobaric UOAs. During wet and dry mode exploration, the former had greater peak areas and signal-to-noise ratios. 61 organic acids of clinical interest were evaluated in patient and laboratory quality control samples. These metabolites were detected to high sensitivity following transition to MRM mode and methyl-malonic acid (MMA) was chosen for initial quantitative analysis. With a R squared of 0.9776 for MMA, this instrument shows strong linearity in the clinical ranges of 10-10000 uM.
**Conclusion:**
The Waters’ Xevo TQ-S micro with Agilent 7890 GC demonstrated promising GC-resolution separation of UOA metabolites coupled to APCI-MS detection. Prior to clinical use, APCI library expansion and further MRM-based quantitative assays need to be done. With clear avenues for future work, the APCI technique offers great benefits for the field of metabolics with LC and GC analysis on a single instrument while improving newborn patient outcome.

Quantitative detection of metabolic urine organic acids via atmospheric pressure chemical ionization gas chromatography mass spectroscopy in multiple-reaction monitoring mode

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