Original

Chromatography, Vol.27 No.3 (2006)

Technical Review

Accurate Mass Measurement on Real Chromatographic Time Scalewith a Single Quadrupole Mass Spectrometer
Yongdong Wang1* and Harry Prest2
12

Cerno Bioscience, Danbury, CT 06810, USA

Agilent Technologies, Santa Clara, CA 95051, USA

Abstract
On a unit mass resolution mass spectrometer such as a single quadrupole MS, it has been widely accepted that the mass (m/z) can only bemeasured to 0.1−0.5 Da accuracy, allowing for only rough mass confirmation for qualitative analysis. A novel mass spectral calibration technique, MSIntegrityTM implemented in MassWorksTM, is introduced to comprehensively calibrate not only the mass axis but also the mass spectral peak shape function. With this comprehensive mass spectral calibration, a high level of mass accuracy on the level of 0.00x Da can beachieved even at unit mass resolution on a true chromatographic time scale, which will now enable the elemental composition determinationof unknown ions or ion fragments using a conventional mass spectrometer hyphenated with chromatographic separation. This identification
capability is further enhanced by a uniquely accurate approach for elemental composition determination through the use of isotope distribution information, CLIPSTM.
Keywords: Accurate mass, mass spectrometry (MS), unit mass resolution, identification, elemental composition, isotope distribution, calibrationof compound identification is a capability typically reserved forIntroduction
Single quadrupole GC/MS has become a workhorse instru-

higher resolution systems such as qTOF or FTMS at a much higher

ment in environmental and other applications due to its reliability,

cost with a larger instrument footprint [4−6]. In order to achieve

cost advantage, ease−of−use, versatility in terms of the types of

the necessary high mass accuracy at the conventional unit masscompounds, high sensitivity, and even portability or at least trans-

resolution, a more elaborate and comprehensive mass spectral cali-

portability [1]. While sufficient for routine applications where abration has to be performed outside the commercially available in-compound is known to belong to a given library, e.g., NIST MS li-

strument systems at the present. Fortunately for GC/MS applica-

brary, these instruments are typically not used for unknown or newtions, such an elaborate calibration is quite feasible by the readily

compound identification, due to their nominally unit mass resolu-available on−board calibration standard, e.g., perfluorotribu-tion and lack of tandem MS capabilities.

tylamine (PFTBA), typically software−controllable through a

As has been shown elsewhere [2−3], even at unit mass resolu-valve. Furthermore, with Electron Impact (EI) ionization widelytion, a high degree of mass accuracy can be achieved, making it

available on GC/MS systems, a molecular ion in many cases ispossible to determine the elemental composition of unknown ionsfragmented into quite a few observable fragment ions which canor fragments and greatly facilitate the identification of metabolites

also be measured with high mass accuracy, providing additional in-or other compounds. Elemental composition search for the purpose

formation for the accurate identification of the molecular ion and1

Cerno Bioscience, Danbury, CT 06810, USA
Agilent Technologies, Santa Clara, CA 95051, USA
TEL: (01) 203−312−1150 FAX: (01) 203−312−1159
E−mail: yongdong.wang@cernobioscience.com
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Chromatography, Vol.27 No.3 (2006)

the profile mode mass spectral scans were repeatedly collected dur-the elucidation of its possible structures.
This short paper will demonstrate such high mass accuracy

ing the GC separation process for a total runtime of 19 min. An

measurement using Agilent 5973 N−inert MSD for the identifica-

elaborate and comprehensive mass spectral calibration can be cre-tion of pesticides through both their molecular ions when available

ated from the average of the PFTBA mass spectral scans within aand their fragment ions, to greatly enhance the capabilities of agiven time window using the MassWorksTM software from Cerno

conventional single quadrupole MS system, which lacks the tan-Bioscience. This unique calibration process calibrates both thedem MS capability and the high resolving power otherwise re-mass and the mass spectral peak shape function, the key for achiev-

quired of highly confident identification work.

ing high mass accuracy. This calibration function was then applied
to each scans in the GC/MS data file to transform each raw mass

Experimentalspectrum into its calibrated version with a mathematically defined

Sample information: Calibration standard PFTBA and a 17−

symmetric peak shape located at accurate mass values. Peak detec-compound organochlorine pesticide standard (1 ng/ul) mixture also

tion can then be applied to reliably and accurately calculate thecontaining approximately 50 ng/ul PCB 209 (decachlorobiphenyl,

mass locations for molecular ions/their fragment ions for the pur-C12Cl10).

pose of compound identification by searching within a small mass

MS conditions: the PFTBA and the mixture were acquired intolerance window for a list of possible formulas. This list of possi-“raw” mode (non−peak detected) at a scan speed 2ˆ2 (A/D samples

ble formulas can be further refined and greatly shortened through= 4) over a mass range of 50−550 m/z.

the Calibrated Line−shape Isotope Profile Search or CLIPSTM, also

Data acquisition and analysis: Figure 1 shows the generalavailable in MassWorks, which utilizes whole isotope profiles toflow of the data processing. The profile mode mass spectra of thedetermine an elemental composition, a highly selective capability

PFTBA calibration standard were acquired continuously for 5 min

made uniquely possible by the comprehensive MS calibration per-during the infusion process while the control valve was at ON posi-formed [7].

tion. Similarly, during the GC/MS analysis of the sample mixture,

Figure 1. The general flow of MassWorks calibration and its elemental composition determination process―１３６―

Chromatography, Vol.27 No.3 (2006)

Yongdong Wang and Harry Prest

Table 1. Calibration Ions from PFTBA Standard and Calibration Mass Errors

IonsIon Formula

Calibration Scans #80−131

Test Scans #684−764

Exact
Monoisotopic
Mass (Da)Calculated Mass(Da)Mass Error(Da)

Calculated Mass(Da)Mass Error(Da)

68.9952

68.99520.000068.9943−0.0009

Frag #1

CF3+

Frag #2

C2 F+
4

99.993699.9931−0.0005

99.9922−0.0014Frag #3

C2F4N+

113.9967

113.9965−0.0002113.9943−0.0024

Frag #4

C2F5+

118.9920

118.9919−0.0001

118.9910−0.0010Frag #5

C3 F+
5130.9920

130.9915−0.0005130.9901−0.0019

Frag #6

C3F7+

168.9888

168.9887−0.0001168.9869−0.0019

Frag #7+
9C4 F

218.9856

218.9858

0.0002218.9847−0.0009

Frag #8+

C5F10N

263.9871

263.9870−0.0001

263.9852−0.0019

Frag #9C7F14N+

363.9807

363.9811

0.0004363.9819

0.0012Frag #10+

C8F16N

413.9775

413.9778

0.0003413.9761−0.0014Frag #11+

C9F18N

463.9743

463.9746

0.0003463.9732−0.0011

Molecular Ion

C9F20N+

501.9711

501.9713

0.0002501.9699−0.0012Figure 2. The raw and the calibrated mass spectrum for one of the calibration ionsbe applied to all the scans to check for the mass accuracy within

Results and Discussion
Twelve ions including the molecular ion of PFTBA are se-this run itself. Once a mass spectral scan has been fully calibrated,lected for the comprehensive MassWorks calibration. Their ele-mass spectral peaks can be accurately determined even for this unit

mental compositions and theoretically calculated exact masses are

mass resolution data. Table 1 lists the calculated masses and mass

listed in Table 1. The average of scans 80−131 is used to build theerrors for all 12 calibration ions for the calibration scans (acquiredcalibration, which transforms the raw mass spectral scan into aearly in the run) as well as for test scans (acquired later in the run).

fully calibrated mass spectral scan, both of which are shown in Fig-

It can be seen that the calibration mass errors are all within 0.5

ure 2 for one of the calibration ions. The calibration thus built canmDa whereas the test mass errors are all within 2.4 mDa.

―１３７―Chromatography, Vol.27 No.3 (2006)

Figure 3. The calibrated mass spectrum for PCB 209 and the accurate masses reported for its five most intense isotopes
Although the results in Table 1 show good mass accuracy on
the calibration ions themselves over a 5 min time period, a more
stringent test would be to apply this calibration to other MS scansfrom a different run, preferably on a true chromatographic timescale. The GC/MS analysis of the pesticide mixture will serve as atrue test of mass spectral calibration, its applicability across different runs and on ions other than the calibration ions on a real chromatographic time scale. Figure 3 shows the accurate masses reported for the average of 8 mass spectral scans corresponding to
the chromatographic elution profile of PCB 209. As can be seen,
the reported accurate masses all come within 4 mDa of the theoretical masses calculated from its elemental composition. While thismolecular ion is known and can be easily verified with certainty,
the identification of some of its EI fragments will be more interesting. For the ion fragment around 424 Da, the accurate mass for themonoisotopic mass is reported as 423.7428 Da, an elemental composition search with C, H, N, O, and Cl as possible elements lists
C12C8+ (exact mass at 423.7503 Da) as the 17th candidate with −7.5
mDa mass error. When the whole isotope distribution is taken into
consideration in CLIPS match, however, C12C8+ becomes the top hit

Figure 4. Isotope matching for the 17 th (top) and the 1 st (bottom) hit from the elemental composition search based
only on monoisotope masseson the list and is indeed the only correct ion formula for this fragment (Figure 4), in spite of its somewhat larger mass measurement
error.
A small chromatographic peak at 12.52 min in Figure 5 is as-monoisotopic mass is reported as 235.0057 Da based on the aver-sociated with a strong ion signal around 235 Da, the accurate

age of 7 scans. An elemental composition search based on this re-―１３８―

Chromatography, Vol.27 No.3 (2006)

Yongdong Wang and Harry Prest

Figure 5. The accurate mass measurement for an EI fragment of pesticide p,p’−DDD

Figure 6. The ion isotope pattern related to the rise in total ion signal towards the end of a GC/MS run(top) and the theoretically calculated isotope patterns of five possible candidate ions (bottom)
deed is the only correct ion formula for a well known EI fragment

ported monisotopic mass with C, H, N, O, and Cl as possible ele+
2ments lists C13H9Clof the pesticide p,p’−DDD.

(exact mass at 235.0081 Da, or −2.4 mDa

mass error) as the 21st hit. The subsequent refinement throughTo demonstrate the application of accurate mass measurement

CLIPS match moves it to the top of the list, revealing that this in-for the identification of unknown compounds, a section corre-―１３９―

Chromatography, Vol.27 No.3 (2006)

sponding to the rise in TIC towards the end of the GC/MS run

GC/MS run on a true chromatographic time scale. The mass shift

shown in Figure 5 is averaged before accurate mass measurement

due to such external calibration is within only a few mDa. The

to help identify possible GC column materials bleeding out of thecomprehensive calibration including the peak shape combined withsystem. Figure 6 (top) shows a section of the averaged mass spec-

CLIPS can greatly enhance the elemental composition determina-trum that is correlated with the rise in TIC signal. With the accuratetion for the purpose of truly unknown compound identification

masses identified, an elemental composition search with possible

from GC/MS experiments.

elements C, H, N, O, and Si combined with CLIPS reveals a few
possible candidates with their theoretical isotope patterns shown inReferences

Figure 6 (bottom). This list of possible candidates can be further re-[1]Troost, J. R.; Olavesen, E. Y. Anal. Chem.; 1996, 68(4) ,708−711.

fined based on the knowledge of column chemistry to improve theunderstanding of column bleeding.

[2]

Gu M, Wang Y, Kuehl D. Spectroscopy, May, 2005.

[3]

Gu M, Wang Y, Zhao X, Gu Z, Rapid Commun. Mass Spectrom. 2006, 20, 764−770.

Conclusions
This application example demonstrates that the comprehen-[4]Blom, K. F. Anal. Chem. 2001, 73, 715.

sive and elaborate mass spectral calibration involving both mass

[5]Tyler, A. et al Anal. Chem. 1996, 68, 3561.

and peak shape is capable of achieving high mass accuracy on a[6]Kind, T. BMC Bioinformatics 2006, 7, 234.

single quadrupole GC/MS system at unit mass resolution. The cali-[7]http://www.cernobioscience.com/resources/CLIPS/CLIPS_Product_Preview.pdf

bration can be conveniently built with the on−board calibration
standard through infusion measurement and is applicable to a real

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