JOURNAL OF MASS SPECTROMETRY, VOL. 30, 381-383 (1995)

JMS Letters Dear Sir,

Polyethylene Glycol: A Reagent for Tuning and Calibrating Mass Spectrometers for Positive-ion Atmospheric Pressure Chemical Ionization

In recent years, atmospheric pressure chemical ionization mass spectrometry (APCI-MS)' has gained enormous popularity2 because of its high sensitivity3 for certain classes of molecules and its ability to be coupled to a liquid chro- m a t ~ g r a p h . ~ On-line liquid chromatography (LC) with an APCI source can be performed at high flow rates, thus making the LC/APCI-MS method an attractive choice for the separation and identification of mixtures using standard ana- lytical columns without the need for flow splitting. The tech- nique, in particular, is found to be extremely powerful for the identification of by-products produced during synthesis, drug metabolites,' impurities and degradation products in drug substances.6 We have found APCI to be a technique of great potential for the routine characterization of synthetic organic molecules. The formation of intense quasi-molecular ions, [M + HI', where M is an analyte of interest, allows the rapid determination of the molecular mass of M.

Despite the utility of APCI-MS and LC/APCI-MS as powerful mass spectrometric tools,'-6 there is no suitable standard available for tuning and calibrating APCI mass spec- trometers. We report here the utility of polyethylene glycols (PEGs) for tuning and calibrating APCI mass spectrometers. PEG has been used previously for the calibration of FAB and electrospray ionization mass spectrometers.' The reagent has several advantages for use in the calibration of mass spec- trometers with an APCI source. First, the mass of the repeat- ing unit of the polymer is 44. Therefore, the instrument can be calibrated with a large number of close calibration data points. This feature is particularly important for sector instru- ments that require close calibration points for good measure- ment accuracy. Second, for precise mass measurement, an unknown ion can be bracketed closely with PEG ions

* Paper prepared for Publication in Organic Muss Spectrometry.

(calibrants) because of the close proximity of PEG ions. Third, PEG is soluble in aqueous solvents containing widely different proportions of organic solvents (e.g. methanol and acetonitrile) commonly used in liquid chromatography. The solubility in the above solvents allows PEGs to be used for tuning and calibration of APCI mass spectrometers without the need to change the LC mobile phase. Finally, when intro- duced into an APCI source with a solvent system containing acetic or formic acid, the most abundant ions produced corre- spond to singly protonated PEG ions undergoing very little fragmentation, resulting in a simple mass spectrum.

An APCI mass spectrum of PEG-2000 is shown in Fig. 1. The most abundant ions in the m/z range 200-2000 arise from the attachment of a single proton to each PEG molecule, H(OCH,CH,)nOH. We used the protonated dimer of meth- anol a t m/z 65.060 as a low-m/z calibration point. The center of the abundance profile of PEG ions, observed at approx- imately m/z 700, is lower than the expected value of approx- imately 2000. This discrepancy may be partly due to the inefficient transmission of high-m/z ions in q ~ a d r u p o l e s . ~ ~ ~ " The low-abundance ions in the m/z range 200-1500 arise from a series of polymers with molecular mass 28 u higher than those of PEG with the same number of -OCH2CH2- repeating units. The abundant ions in the m/z range 50-200 (m/z 89, 133 and 177) correspond to another series of polymers whose molecular mass is 18 u lower than those of PEG mol- ecules with the same number of repeating units. The origin of these two series of ions is being investigated.

We also studied PEG-400, PEG-1000 and polypropylene glycol (PPG)-1000 and PPG-2000 for calibration of an APCI mass spectrometer. The abundance of ions in the high-m/z region (> 1400) for the first three polymers is low. The PPG- 2000 mass spectrum yields a similar intensity profile to that of PEG-2000. Using PEG ions, the quadrupole mass spectrom- eter was tuned in APCI using an auto-tune procedure (see below). Several prameters used in the present investigation are shown in Fig. 2 (see also the caption of Fig. 1). The higher value for the peak width of half-height for high-m/z ions

100

8 0

.+ 60 v) C al C u -

4 0

2 0

1:

1 1x2

6 : 6 3 5 . 6 1

5 3 1 . 4 1 1

.1 5 4 7 . 5

500

5 E+ C7 1x40 I

7 p . 7

7 6 2 . 0

8 1 1 . 7

1428,. 1

I

1 0 0 0

mlz 1 5 0 0 2 0 0

1.22

Figure 1. APCI mass spectrum of PEG-2000. The flow rate of mobile phase (methanol-water (70: 30). 3% acetic acid) was 500 pI min-'. The PEG solution was added to the mobile phase through a T-junction to a final concentration of 100 PM, The APCI conditions were nebulizer temperature = 520°C. capillary tube temperature 230°C. auxiliary gas flow rate = 20 cm3 min-', sheath gas pressure = 50 psi and corona current = 6 FA. Scan range, m/z 50-2000; averaging time, 2 min. Note the magnification of the intensity scale: x2 for rn/z 250-1450 and x40 for mlz 1450-2000.

CCC 1076-51 74/95/020381-03 0 1995 by John Wiley & Sons, Ltd.

Received 14 July 1994 Revised I August 1994

382

Auto-tune mass list - U S E R 2

LETTERS

Exact m/z Peak width (1/2 height)

1 = 65.06 0.8

2 = 239.149 0.8 3 = 679.411 0.8

4 = 899.543 0.8

5 = 1339.804. 1.1 6 = 1647.988 1.2

7 = 1868.119 1.3

Tune Lab18 5. tune flle tune09 from 28 JUN 9 4 9 23 CdlibrbLlon 56.61

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U T) 49.98

3 1 4 5 5 1 7 6 . 7 9 8 I

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Figure 2. Tuning parameters used in the present investigation. The figure is from screen dumps obtained from Finnigan SSQ-710 during calibration. The upper trace shows the mass list and peak width. The bottom left trace shows the calibration graph and the bottom right trace the output of the calibration DAC value and the peak profile of the highest m/z ion (1868.119). The DAC values are assigned to different m/z during calibration.

(> 1300) is required to offset the poor transmission of high-m/ z ions in quadrupoles. Even for the highest m/z ion (1868) used for tuning and calibration, the isotopic cluster is sufficiently resolved for calibration with monoisotopic m/z values (Fig. 2). To our knowledge, most of the applications of APCI require calibration only up to m/z 1500. A complete set of m/z values of protonated PEG up to m/z 2000 is given in Table 1.

The data presented in Fig. 1 were acquired on a Finnigan SSQ-710 (Finnigan Mat, San Jose, CA, USA) single- quadrupole mass spectrometer equipped with a Finnigan APCI source and a - 20 keV conversion dynode for enhanced sensitivity. A 10 mM PEG-2000 (Aldrich, Milwaukee, WI,

USA) solution was prepared in a 1 : 1 mixture of methanol (EM Science, Gibbstown, NJ, USA) and distilled water (prepared with a Millipore Milli-Q distillation apparatus). A solvent mixture consisting of 70% methanol and 30% distilled water, each containing 3% acetic acid (J. T. Baker, Phil- lipsburg, NJ, USA), was delivered at a flow rate of 500 p1 rnin-' to the mass spectrometer by a Waters 600MS HPLC pump system (Waters, Milford, MA, USA). The PEG solution was added to the flowing solvent mixture prior to entering the APCI source through a T-junction at a flow rate of 5 p1 rnin-' using a Harvard Apparatus (South Natick, MA, USA) Model 22 syringe pump. The final concentration of the PEG

Table 1. Calculated monoisotopic m/z values corresponding to protonated PEG ions, [ HO(CH,CH,O)nH]H+, n = 1-45

n mi2

1 63.044605 2 107.070819 3 151.097034 4 195.123249 5 239.149464 6 283.175679 7 327.201 893 8 371.228108 9 415.254323

10 459.280 538 11 503.306 753 12 547.332 967 13 591.359 182 14 635.385 397 15 679.41 1 61 2

n mi2

16 723.437 827 17 767.464 041 18 81 1.490 256 19 855.51 6471 20 899.542 686 21 943.568 901 22 987.595 11 5 23 1031.621 330 24 1075.647 545 25 11 19.673 760 26 11 63.699 975 27 1207.726 189 28 1251.752 404 29 1295.778 61 9 30 1 339.804 834

n mi2

31 1383.831 049 32 1427.857 263 33 1471.883 478 34 1 51 5.909 693 35 1559.935 908 36 1603.962 123 37 1647.988 337 38 1692.01 4 552 39 1736.040 767 40 1780.066 982 41 1824.093 197 42 1868.119411 43 191 2.1 45 626 44 1956.1 71 841 45 2000.1 98 056

LETTERS 383

in the mobile phase was 100 p ~ . The mass spectrometer was scanned from m/z 50-2000 in 2 s in the profile mode and the final spectrum was averaged for 2 min. The mass spectrum presented in Fig. 1 was acquired following tuning and cali- bration using the experimental conditions described above. The tuning was accomplished using the Finnigan Auto-tune procedure from the Guide view of the Finnigan ISIS software.

The m/z values shown on the ion peaks in Fig. 1 are mea- sured values. The average deviation of the measured m/z values from those calculated (Table I), derived from the most intense 36 peaks in Fig. 1, is 0.15 0.1. The typical accuracy obtained in the molecular mass determination in APCI is k0.2 u. In our experience, the calibration holds for at least 2 months unless the mass spectrometer is dismantled for clean- ing.

Although the tuning and calibration method presented here were investigated on a quadrupole mass spectrometer, the application is by no means limited to this instrumentation alone and should be applicable to tune and calibrate effec- tively other systems such as sectors, ion traps time-of-flight and ion cyclotron resonance mass spectrometers for APCI.

We thank Drs R. Weinkam and D. Johnston for their support.

Yours

HUNG-YU LIN, GEORGE J. GONYEA and SWAPAN K . CHOWDHURY* Analytical Sciences Department, Sterling Winthrop Pharmaceutical Division, 1250 S. Collegeville Road, Collegeville, PA 19426, USA

References 1.

2.

3.

4.

5

6

7

(a) E. C. Horning, M. G. Horning, D. I. Carrol, I. Dzidic and R. N. Stillwell, Anal. Chem. 45, 936 (1973); (b) D. I . Carrol, I. Dzidic. E. C. Horning and R. N. Stillwell, Appl. Spectrosc. Rev. 17, 337 (1981). (a) R. D. Voyksner, Environ. Sci. Techno/. 28, 118A (1994); (b) A. P. Bruins, Trends Anal. Chem. 13, 37 (1994); (c) E. C. Huang, T. Wachs, J. J. Conboy and J. D. Henion, Anal. Chem. 62, 71 3A (1 990). (a) B. A. Thompson, T. Sakuma, J. Fulford, D. A. Lane, N. M. Reid and J. B. French, Adv. Mass Specfrom. 88. 1422 (1 980); (b) J. Sunner, G. Nicol and P. Kebarle, Anal. Chem. 60, 1300 (1988); (c) J. Sunner, M. G. Ikonomou, and P. Kebarle, Anal. Chem. 60, 1308 (1988); (d) M. Sakairi and H. Kambara, Anal. Chern. 60,774 ( I 988). (a) T. Covey, E. Lee, A. Bruins and J. D. Henion, Anal. Chem. 58, 1451A (1986); (b) M. G. Ikonomou, A. Naghipur, W. J. Lown and P. Kebarle, Biomed. Environ. Mass Spectrom. 19, 434 (1990); (c) D. Thomas, P. G. Sim and F. M. Benoit, Rapid. Commun. Mass Scpectrorn. 8, 105 (1994); (d) L. N. Tyreforos, R. X. Moulder and K. E. Markides. Anal. Chem. 65, 2835 (1 993). (a) T. Covey and B. Shushan, Nippon lyo Masu Supekutoru Gakkai Koenshu 16, 27 (1991); (b) P. V. Macrae, B. C. Jones and J. F. J. Todd, Rapid Comrnun. Mass Spectrom. 7 , 605, 1993. (a) I. M. Johansson, R. Pavelka and J . D. Henion, J. Chro- mafogr. 559, 515 (1991); (b) X.-2. Qin. D. P. Ip, K. H.-C. Cheng, P. M. Dradransky, M. A. Brooks and T. Sakuma, J. Pharm. Biomed. Anal. 12, 221 (1 994). (a) K. J. Bare, Org. Mass Specfrom. 20, 693 (1985); (b) L. J. Goad, M. C. Prescott and M. E. Rose, Org. Mass Spectrom. 19, 101 (1984); (c) R. B. Cody, J. Tamura and B. D. Musselman, Anal. Chem. 64, 1561 (1992).