A Proposed Strategy for the Detection of Metabolic Disorders:High Resolution Screening with Reflex to Targeted Quantitation by Tandem Mass Spectrometry
Julie A. Ray1, Heather Golsan1, Natalie Rasmussen1, Frederick G. Strathmann1, 2
1ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT, 84108, United States
2Department of Pathology, University of Utah, Salt Lake City, UT, 84108, United States•••Sample selection: 50 human plasma samples with abnormallyhigh levels of Phe (196-1263 µmol/L; age range 0-64 years) and10 samples with normal concentrations of Phe (45-80 µmol/L; age
range 0-43 years).
Sample preparation: 100 µL of the samples were subjected toprotein precipitation using equal volume of 0.4N HClO4, shaken
and allowed to sit for 15 min at room temperature and filteredusing AcroPrep Advance 0.2 µm 94 well filter plates (PALL LifeSciences USA).
Abnormal samples were diluted 10, 20, 30, 40 and 50 times;
normal samples were diluted 30 times using 0.1N HCl before
being subjected to LC-QTOF-MS analysis. To account fortechnical variation each normal sample was injected twice. Areacounts of serially diluted abnormally high Phe samples werecompared with concentrations of samples measured by aquantitative LC-MS/MS assay ((API 4000 mass spectrometer;using an 18 min HPLC-MS/MS aTRaqTM reagent method).
Data analysis: Data were analyzed with Agilent Mass Hunter
Qualitative Analysis (Qual) B.07.00 software using the find byformula search algorithm, comparing results to a customized
database containing the analyte of interest. Exact mass match,isotope pattern and spacing, and retention time match were usedfor identification.

MS Parameters (Agilent 6550 TOFSystem)•) Ion Polarity
Positive••
•
•••
•
•••
•
••

Gas temperature
Drying gas
Sheath gasNebulizer
Sheath gas temperature
Capillary voltage
Nozzle voltage
MS range
Acquisition rate
MS/MS range
Collision energy
Reference mass
922.0098
Reference pump flow rate

•
••
•
•

250oC
15 L/min12 L/min
45 psi
400oC
3500V
0V
50-1000 m/z
1 spectra/sec
NANA
121.0509, 149.0233, 322.048,
0.500 mL/min

Area count LC-TOF-MSµm (Imtakt USA)
Guard column
Pre column 2 µm filter
Column temp
350oC
Autosampler temp4oCMobile phase
A: 100mM ammonium formate; B: 0.1%formic acid in acetonitrile
LC conditions (10 min) 0-3 min 14% A, 3.1-10 min 100% A
LC conditions (1 min) 0-1min 14% A, 1.1-1.5 min 100% A
Flow rate
0.6mL/min
Stop time
10 minPost time
0.5min•••Phe (C9H11NO2) eluted at 2.56 and 0.92 min using the 10 and 1min gradient methods respectively. It was detected within a mass
and retention time tolerance of ±15 ppm and ±0.05 min in Mass
Hunter Qualitative Analysis using Find by Formula search in thespecified database (Fig 1A, B, C).
Linearity of response of serially diluted abnormal Phe samples wasfound between 20 and 40 factors of dilution with correlation
coefficients being 0.77 and 0.89 respectively (Fig 2).
Comparison of corresponding area counts for Phe for 2 and 40 folddilution on the LC-QTOF-MS using the 1 and 10 min gradientmethods vs concentrations measured by the quantitative LCMS/MS method showed acceptable correlation (Fig 3A,B).
The LC-TOF-MS method was able to detect DL-βaminoisobutyric acid (C4H9NO2, retention time 4.66 min, masstolerance ±17 ppm and retention time tolerance ± 0.05 min) in all
samples while the quantitative LC-MS/MS method failed to detect
the analyte in 30% of the normal samples (Fig 4).

Figure 1A: Phe in normal patient sample (10 min method)

2E+09

1E+09

Dil x10R2= 0.77

Dilx 20

R2= 0.76

Dil x30

R2= 0.85

Dil x40

R2= 0.89

Dil x50

R2= 0.78

00500

1000
Phe (µmol/L) LC-MS/MS1500

Figure 3A: Correlation between area counts of LC-TOF-MS (1 minmethod) and concentration of Phe using LC- MS/MS; LC-QTOF-MS =1.30e5 LC-MS/MS + 1.26e7, n= 50, r = 0.88, Sy/x = 98.52

Results•Methods•••••Figure 2: Area count (LC-TOF-MS) in patient samples diluted by 10-50
factors containing abnormally high concentrations of Phe vsconcentration of Phe using LC-MS/MS

Area count LC-TOF-MS, dilution
factor 2, 1min gradient method

Amino acids are essential for every metabolic process. Amino acidanalysis is required for the diagnoses and screening of primary andsecondary aminoacidopathies as well as for the monitoring oftherapeutic responses and assessment of nutritional status.
Phenylketonuria (PKU) is characterized by accumulation of
Phenylalanine (Phe) in the blood due to mutations in the Phehydroxylase gene, resulting in low levels of the enzyme Phehydroxylase which prevents the conversion of excess Phe to tyrosine.
Untreated PKU can lead to seizures and intellectual disabilities.
Considering the complexity required for sample preparation,
separation of analytes of interest from interferences and required
detection levels with existing LC-MS/MS methods in amino acidanalysis, we explored the possibility of using a LC-QTOF-MS system
run in TOF mode as a higher throughput, simplified, qualitative screen
for the detection of metabolic disorders such as PKU in humanplasma. The results were analyzed using the Agilent Mass Hunter
Qualitative Analysis and compared with an existing quantitative LCMS/MS method for amino acid measurement.

LC Parameters (Agilent 1290 Infinity LCsystem)•) Analytical column
Intrada Amino Acid 50 mm x 3 mm x 3

3.E+082.E+081.E+080.E+00
0500

1000

1500

Phe (µmol/L), LC-MS/MSFigure 3B: Correlation between area counts of LC-TOF-MS (10 minmethod) and concentration of Phe using LC-MS/MS; LC-QTOF-MS =1.0e6 LC-MS/MS -1.30e8, n= 50, r = 0.89, Sy/x = 92.43
Area count LC-TOF-MS, dilution
factor 40, 10 min gradient method

Introduction

2E+09

1E+09

00500
1000
Phe (µmol/L) LC-MS/MS

1500

Figure 4: DL-β-aminoisobutyric acid detected at 4.66 min by LC-TOFMS in normal patient sample

Conclusion
Figure 1B: Phe in abnormal patient sample (10 min method)•••

Figure 1C: Phe in abnormal patient sample (1 min method)

The described LC-TOF-MS method is characterized by ease ofwork flow when compared with the LC-MS/MS method.
There is acceptable agreement between the two methods. Due tothe high mass accuracy of the QTOF, the screening techniqueenables easy method set up without precursor ion selection andfragmentation conditions.
Use of Q-TOF platform as the screen allows the retrospective
interrogation of collected MS without re-injection of sample.

Acknowledgements
We thank ARUP Institute for Clinical and Experimental Pathology®
for supporting this project. We are grateful to Julie Cichelli andDaniel Cuthbertson from Agilent Technologies, Inc. and Robert
Puryear from Imtakt USA for their help.