Determination of phenylalanine isotope ratio ...

Z. Wu et al., Eur. J. Mass Spectrom. 10, 619–623 (2004)

619

Determination of phenylalanine isotope ratio enrichment by liquid chromatography/ time-of-flight mass spectrometry

Zhanpin Wu,a* Xiao-Jun Zhang,b Robert B. Codya and Robert R. Wolfeb a

Analytical Instrument Division, Jeol USA, Inc., 11 Dearborn Road, Peabody, MA 01960, USA. E-mail: [email protected]

b

Metabolism Division, Department of Surgery, University of Texas Medical Branch at Galveston, Galveston, TX 77550, USA

The application of time-of-flight mass spectrometry to isotope ratio measurements has been limited by the relatively low dynamic range of the time-to-digital converter detectors available on commercial liquid chromatography/time-of-flight mass spectrometry (LC/ToF-MS) systems. Here, we report the measurement of phenylalanine isotope ratio enrichment by using a new LC/ToF-MS system with wide dynamic range. Underivatized phenylalanine was injected onto a C18 column directly with 0.1% formic acid/ acetonitrile as the mobile phase. The optimal instrument parameters for the time-of-flight mass spectrometer were determined by tuning the instrument with a phenylalanine standard. The accuracy of the isotope enrichment measurement was determined by the injection of standard solutions with known isotope ratios ranging from 0.02% to 9.2%. A plot of the results against the theoretical values gave a linear curve with R2 of 0.9999. The coefficient of variation for the isotope ratio measurement was below 2%. The method is simple, rapid and accurate and presents an attractive alternative to traditional gas chromatography/mass spectrometry applications. Keywords: time-of-flight mass spectrometry, isotope ratio enrichments, amino acids

Introduction Measurement of phenylalanine isotope ratio enrichment is critical to metabolic studies.1 Gas chromatography/ mass spectrometry (GC/MS)2,3 and/or (GC-combustion) isotope ratio mass spectrometry (IRMS)4 are commonly used for the measurement. Unfortunately, a time-consuming derivatization procedure is required. An isotopic enrichment calibration curve is also generally needed for the enrichment below 0.1% if GC/MS is used. The derivatization also dilutes the isotope label for GC-combustion/IRMS measurements so that a higher tracer infusion rate and a longer period experiment are needed to achieve measurable enrichments in metabolic studies. Some LC/MS methods5,6 have also been reported. However, the use of time-of-flight mass spectrometry for isotope ratio measurement has not been reported. In contrast to conventional scanning instruments (for example, quadrupole and magnetic sector mass spectrometers) that must scan over one mass at a time, time-of-flight (ToF) mass spectrometers detect ions simultaneously across

DOI: 10.1255/ejms.670

the full mass range. This may reduce the effect of temporal variations in the ion source on the measured isotope ratios. A very accurate peak area measurement for all isotopomers can be achieved. However, the dynamic range is relatively low in ToF-MS if a TDC (time-to-digital converter) is used as a data acquisition system.7 The disadvantage of narrow dynamic range hinders the applications for isotope ratio enrichment measurement. Recently, a new LC/ToF-MS system was introduced that achieves a wide dynamic range by using an ADC (analog-to-digital converter) instead of a conventional TDC as a data acquisition system.8 We used this system to measure phenylalanine isotope ratio enrichment. 13C6-labeled phenylalanine standard was used to evaluate the sensitivity, reproducibility and concentration dependency. A comparison between this method and the traditional GC/MS method was also made in rabbit skin protein samples after 2H5-phenylalanine was infused. All standards and samples were injected into the LC/MS system directly without derivatization. The method is simple, rapid and accurate and presents an attractive alternative to traditional GC/MS applications.

ISSN 1356-1049

© IM Publications 2004

620

Experimental Chemicals and preparation of standard solutions

All solvents used were of HPLC grade. The standard solutions were prepared by mixing unlabeled phenylalanine (Sigma, Saint Louis, MO, USA) and 13C6-phenylalanine (Cambridge Isotope Laboratory, Inc., Andover, MA, USA) in 0.1% formic acid solution to make solutions with isotope ratio enrichments ranging from 0.02% to 9.2% and a constant total concentration of 100 nmol mL–1. Biological tracer studies

Biological samples were derived from a conscious rabbit study,9 in which 2H5-phenylalanine was infused into a preinstalled catheter in the jugular vein at 0.2 µmol kg–1 min–1 (prime: 8 µmol kg–1). After 8 h primed-constant infusion, skin samples were taken from the back under general anesthesia of ketamine and xylazine. Skin sample processing

The skin samples were processed for protein-bound enrichment measurement as described previously.1 Briefly, approximately 50 mg of each sample was homogenized three times in 5% perchloric acid solution at 4°C and the protein precipitate was washed sequentially three times with 2% perchloric acid solution, twice with absolute ethanol and once with ether to remove free amino acids and lipids. The washed precipitate was dried overnight in an oven at 80°C and hydrolyzed in 6 N HCl for 24 h at 110°C. The protein hydrolysate was then passed through cation–ion exchange columns and eluted with 2 N NH4OH. The resulting eluate, which contained amino acids including phenylalanine, was collected and divided into two aliquots. After drying under vacuum in a Speed-Vac rotary drying apparatus (Savant Instruments, Farmingdale, NY, USA), one aliquot was reconstituted in 500 µL of 0.1% formic acid solution for LC/MS analysis and the other aliquot was used to make the t-butyldimethylsilyl (t-BDMS) derivative of phenylalanine for GC/MS analysis, as described previously.10 Briefly, 100 µL of N-methyl-N-(tert-butyldimethysilyl) trifluoroacetamide (MTBSTFA; Pierce, Rockford, IL, USA) and 100 µL of acetonitrile were added to the dry samples. The mixture was then heated at 95°C for 45 min to produce a derivative of phenylalanine. GC/MS analysis

Phenylalanine enrichment in the t-BDMS derivatives was measured on a Hewlett-Packard 6890/5973 GC/MSD with a 30 m × 0.25 mm × 0.25 µm HP-5MS capillary column (GC conditions: initial temperature 70°C, held for 0.5 min; slow ramp 11°C min–1 to 240°C, held for 0.1 min; rapid ramp 50°C min–1 to 280°C, held for 1 min). Mass spectrometry was performed using electron ionization (EI) with selected ion monitoring (SIM) at m/z 234, 237 and 239 (M + 0, M + 3, and M + 5 isotopomer ions, respectively). The ion source temperature and the transfer line temperature were 230°C and 280°C, respectively. The electron multiplier voltage

Determination of Phenylalanine Isotope Enrichment

was set to 1529 V. The data recording cycle time was 4.35 cycles s–1 for all three selected ions. The mass spectrometer was tuned using the Auto Tune method. The enrichment of m/z 239/237 (M + 5/M + 3) was converted to the enrichment of m/z 239/234 (M + 5/M + 0) using a standard calibration curve, which was derived from a set of standard samples with known 2H5-phenylalanine enrichment. LC/ToF-MS analysis

An Agilent 1100 HPLC system (Wilmington, DE, USA) was used. All standard solutions and samples were injected onto a 2.0 × 150 mm, 5 µm particle, Luna C18 (2) column (Phenomenex, Torrance, CA, USA). The injection volume was 10 µL. A linear gradient elution from 100% mobile phase A (0.1% formic acid) to 12% mobile phase B (100% acetonitrile) in 12 min was performed at a constant flow rate of 0.2 mL min–1. The mass spectrometer system consisted of a JEOL AccuToF time-of-flight (ToF) mass spectrometer with standard electrospray ion source (ESI) and a JEOL MassCenter workstation (JEOL USA, Inc, Peabody, MA, USA). The system was set to positive-ion mode and optimized by using a syringe pump to infuse a phenylalanine standard solution at a flow rate of 0.2 mL min–1. The mass spectrometer was tuned to maximize sensitivity at a resolving power of 5000 (FWHM). The needle voltage and orifice 1 voltage were set to 2200 V and 35 V, respectively. These potentials minimized the phenylalanine solvent adduct ions and maximized the peak for protonated phenylalanine. The microchannel plate (MCP) voltage was set to 2700 V. The desolvating gas flow rate was set to 2.5 L min–1 and the nebulizing gas flow rate was set to 1.0 L min–1. The temperatures for the desolvation chamber and orifice 1 were set to 200°C and 80°C, respectively. High-resolution mass chromatograms were generated for the measurement of isotopomers peak area. Determination of TTR

The isotope enrichment was expressed as tracer/tracee ratio (TTR). The TTR was calculated by getting the isotopomer peak areas for phenylalanine in high-resolution mass chromatograms. The ratio of m/z 171/166 (M + 5/M + 0) was used for 2H5-phenylalanine enrichment calculation; the ratio of m/z 172/166 (M + 6/M + 0) was used for 6C13-phenlylalanine enrichment calculation, where M is the monoisotopic mass of protonated phenylalanine ion. Results and discussion LC/MS measurements of isotope ratios require not only the determination of the areas of the mass chromatographic peak for the spectrum’s base peak but also of the mass chromatograms of the isotopomeric peaks, which have a much lower abundance. In this study, the samples were injected into the system directly without derivatization. A high-resolution mass chromatogram is required to


x10

3

621

Intensity (161821) x1

161.80

1.781

x1

a

161.60

11.531

SN:22.4

161.40

161.20

161.00

x10

3

Intensity (161532) x1

x1

11.531 SN:36.9

b

161.40

161.20

161.00

0

2

4

6

Time[min]

8

10

12

14

2

Figure 1. Mass chromatograms for a low-level (0.07%) 2H5-phenylalanine extracted from rabbit skin protein, (a) low-resolution m/z 171 ± 0.5, (b) high-resolution for m/z 171.118 ± 0.03.

10 9 8 7 6 5 4 3 2 1 0

y = 1.0188x + 0.0129 R2 = 0.9999

alanine extracted from rabbit skin protein. Figure 1(a) shows the mass chromatogram constructed for m/z 171 ± 0.5 (“low resolution mass chromatogram”) and Figure 1(b) shows the mass chromatogram constructed for m/z 171.118 ± 0.03 (“high resolution mass chromatogram”). A comparison of the two mass chromatograms shows that the signal-to-noise ratio increases from 22 for the low-resolution mass chromatogram to 36.9 for the high-resolution mass chromatogram. In addition, the abundance of an early-eluting interference peak is significantly reduced in the high-resolution mass chromatogram. The instrumental detection limits and linearity were determined for the 13C6-labeled phenylalanine standards. Peak areas were calculated from the high-resolution mass

1.75 1.5 1.25

TTR (%)

Measured TTR

obtain accurate peak areas and avoid possible problems with coeluting interferences or background peaks. Therefore, the system should be tuned to achieve high sensitivity and high resolution. Under the tuning conditions, a resolution of 5000 (FWHM) was achieved and ESI produced only protonated molecules of phenylalanine with a very small amount of acetonitrile adduct ions. There was no fragmentation. This enables high detection sensitivity. Figure 1 shows the mass chromatograms for m/z 171 (M + 5 ion, where M is the monoisotopic mass of protonated phenylalanine ion) for a low level (0.07%) deuterium-enriched (2H5-) sample of phenyl-

1 0.75 0.5 0.25

0

2

4

6

8

10

Theoretical TTR

Figure 2. Relation between the theoretical ratios and the measured ratios.

0 0

0.2

0.4

0.6

0.8

1

1.2

nmol injected

Figure 3. Relation between the injected amount and isotope ratio measurement.

GC/MS TTR (%)

622

Determination of Phenylalanine Isotope Enrichment

0.45 0.4 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0

y = 1.0034x + 0.0228 R2 = 0.9583

0

0.1

0.2

0.3

0.4

LC/MS TTR (%)

Figure 4. Correlation between measurements of phenylalanine enrichment by LC/MS and GC/MS.

chromatograms for M + 0 and M + 6 isotopomers (M is the monoisotopic mass of protonated phenylalanine ion). To determine the accuracy of the isotope ratio measurement, a plot of the measured results against the theoretical values was constructed and is shown in Figure 2. The enrichment range was from 0.02% to 9.2%. A linear correlation was found with an R2 of 0.9999. The slope was determined to be 1.0188 and the y-intercept was 0.0129, indicating excellent accuracy. Below 0.05% enrichment, the precision for M + 6 peak areas became more variable. Therefore, we consider 0.05% to be the enrichment detection limit of this method. The reproducibility of the method was determined from five injections of a standard solution with 0.94% enrichment. The relative standard deviation (RSD) for the measurement was 1.9%, indicating a good reproducibility. There is a report that isotope ratio enrichment measurements for many GC/MS applications are affected by the concentrations of sample analyzed.11 In order to determine this concentration dependency, a standard phenylalanine solution with 0.94% enrichment was diluted to concentrations ranging from 1.1 to 106.0 nmol mL–1 and 10 µL of each solution was injected into the system. In Figure 3, the relation between the injected amount and the isotopic ratio is shown. The measured ratios were independent of the amount of phenylalanine analyzed over the range of 0.03 to 1 nmol. Hence, the lowest concentration required to get an accurate enrichment measurement is 3 nmol mL–1 and the measured isotope ratio enrichments are independent over a wide concentration range. A comparison of phenylalanine enrichments obtained by this method with the values obtained by traditional GC/ MS is shown in Figure 4 for rabbit skin protein after a 2H5phenylalanine isotope tracer infusion experiment. There was excellent agreement between the two methods, with a slope of 1.0034, a y-intercept of 0.0228 and an R2 of 0.9583. For isotope enrichment measurements, time-of-flight mass spectrometers have several potential advantages over scanning mass analyzers (quadrupole and magnetic sector mass spectrometers). Because the resolving power does not depend on slit widths or position on a stability diagram, high resolving power is achieved with no loss in signal. This provides high sensitivity (required for accurate peak area measurements and the detection of low-level isotopomers) and high resolving

power (required to eliminate background interferences) within the same measurement. Scanning mass spectrometers generally use “peak-hopping” (selected ion monitoring) to achieve the best sensitivity and quantitative accuracy. Temporal variations in the ion source during peak-hopping can reduce the accuracy and precision of measurements made with scanning mass spectrometers. Time-of-flight mass spectrometers measure isotopomers that are extracted from the ion source at the same instant. This may reduce the effect of temporal variations in the ion source on the measured isotope ratios. These benefits of time-of-flight for isotope enrichment measurements can only be achieved by using a detector with sufficient dynamic range to allow accurate measurements of isotopomers with widely varying abundances. In this report, we have shown that a time-of-flight mass spectrometer, using a continuous averager detection system, can provide accurate and precise measurements of isotopic enrichment. The method we developed on this LC/MS system offers several advantages over traditional GC/MS. The samples can be injected directly without a tedious derivatization procedure. The total run time for each sample is only 12 min. Under optimized MS conditions, there were no fragment ions presented in the mass spectrum (spectrum not shown). The most intense isotopomeric ions can be detected, enabling highly accurate peak area determination. The method can be used to measure as low as 0.05% enrichment without using an isotopic enrichment calibration curve. The measured enrichment is independent over a wide concentration range. Conclusion An LC/ToF-MS system with an ADC as the data acquisition system can be used to measure isotope ratio enrichment. The method is simple, rapid and accurate and presents an attractive alternative to traditional GC/MS applications. Acknowledgment The authors wish to thank Yuxia Lin and Gaurang Jariwala for expert technical assistance. References 1. R.R. Wolfe, Radioactive and Stable Isotope Tracers in Biomedicine: Principles and Practice of Kinetic Analysis. John Wiley & Sons, Inc., New York, USA (1992). 2. B.W. Patterson, X.-J. Zhang, Y.P. Chen, S. Klein and R.R. Wolfe, “Measurement of very low stable isotope enrichments by gas chromatography/mass spectrometry: application to measurement of muscle protein synthesis”, Metabolism 46, 943 (1997). 3. H. Schweer, B. Watzer, H.W. Seyberth, A. Steinmetz and J.R. Schaefer, “Determination of isotopic ratios of L-leucine and


4.

5.

6.

7.

L-phenylalanine and their stable isotope labeled analogues in biological samples by gas chromatography/triple-stage quadrupole mass spectrometry”, J. Mass Spectrom. 31, 727 (1996). K.E. Yarasheski, K. Smith, M.J. Rennie and D.M. Bier, “Measurement of muscle protein factional synthetic rate by capillary gas chromatography/combustion isotope ratio mass spectrometry”, Biol. Mass Spectrom. 21, 486 (1992). H.M.H. Van Eijk, D.R. Rooyakkers, P.B. Soeters and N.E.P. Deutz, “Determination of amino acid isotope enrichment using liquid chromatography-mass spectrometry”, Anal. Biochem. 271, 8 (1999). C. Papageorgopoulos, K. Caldwell, C. Shackleton, H. Schweingrubber and M.K. Hellerstein, “Measuring protein synthesis by mass isotopomer distribution analysis (MIDA)”, Anal. Biochem. 267, 1 (1999). I. Chernushevich, A. Loboda and B. Thomson, “An introduction to quadrupole-time-of-flight mass spectrometry”, J. Mass Spectrom. 36, 849 (2001).

623

8. J. Tamura and J. Osuga, “New generation LC-ToF/MS AccuToF™”, J. JEOL News. Analytical Instrumentation 28A, 11 (2002). 9. X.-J. Zhang, Y. Sakurai and R.R. Wolfe, “An animal model for measurement of protein metabolism in the skin”, Surgery 119, 326 (1996). 10. A.A. Ferrando, H.W. Lane, C.A. Stuart, J. Davis-Street and R.R. Wolfe, “Prolonged bed rest decreases skeletal muscle and whole body protein synthesis”, Am. J. Physiol. Endocrinol. Metab. 270, E627 (1996). 11. B.W. Patterson, G. Zhao and S. Klein, “Improved accuracy and precision of gas chromatography/mass spectrometry measurements for metabolic tracers”, Metabolism 47, 706 (1998).

Received: 27 April 2004 Revised: 3 June 2004 Accepted: 7 June 2004 Web Publication: 21 September 2004