New developments in quantitative mass spectrometry

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7 Beveridge, A. D. et al. (1975) J. Forensic Sci. 20, 43 1 8 Solon, J., Watkins, J. and Mikkelsen, H. (1971) Automated Analysis of Alcohols in Blood, Hewlett-Packard Co., Avondale, PA 9 Doyle, T. D., Herine, J. (1978) J. Assoc. 08 Anal. Chem. 61, 172 10 Horowitz, W. (ed.) Ojkial Methoa? of Analysis of the Association of OfjScial Analylical Chemists, 13th edn, Association of Official Analytical Chemists, Washington, D.C.

11 The Pharmacopeia of the United States of America (XVIII Rev) (1970) U.S. Pharmacopeial Easton PA

Convention,

Inc., Mack Publishing Co.,

12 Harbin, D. A. and Lott, P. F. ( 1980) J. Lig. Chromatog. 3, 243

. Dennis J. Dams received his B.S. in Chemistryfrom the University of Mirsouri-Kansas Cig in 1976. He is now supervisor of the Analytical Chemistry andlnstrumentation Section of the Kansas Cio Regional Criminalistics Laboratory (2100 N. Noland Road, Independence, MO 64050, U.S.A .) where he is involved with the analysis offorensic samples such as pharmaceuticals, poisons, explosives, etc. and with the im@mentation, operation and the maintenance of laboratory instrumentation. Peter F. Lott received his B.S. and M.S. degreesfiom St. Lawrence University and his Ph.D. from the University of Connecticut in 1956. His present position is as Professor of Chemistry at the Universityof Missouri-Kansas Cig, MO 64110, U.S.A.

New developments in quantitative spectrometry

mass

Sensitivity and specificity are the ma’or advantages of mass spectrometry as a quantitative ana I ytical technique. Both may be enhanced by the use of recently developed methods of ion production and characterization.

Simon J. Gaskell

Selected ion monitoring

Cardiff, U.K.

This review will concentrate on those areas where the analytical requirement is for reliable quantification of one or a few trace components present in complex mixtures. In general, this implies the use of some form of selected ion monitoring (SIM), i.e. the selective detection of a single ion, or a few ions characteristic of the components of interest. Quantitative analyses using repetitive mass scanning will not be considered here; with this technique, quantification of all major components may be achieved by post-analysis reconstruction of mass chromatograms (selected ion profiles). While the advantages of this approach in multi-component analyses are clear, sensitivity is reduced by comparison with SIM and high precision is generally not achieved (though it may be adequate for many applications). The rapid development of quantitative MS may be traced to the first experiments employing SIMlp*, While the technique is commonly associated with combined gas chromatography-mass spectrometry (GC-MS), it is of course applicable to all means of sample introduction. It is, perhaps, unfortunate that the great selectivity of the approach in terms of detection has obscured the non-selectivity of compound ident@cation that it may represent. Frequently, during SIM analyses, the vast majority of mass spectrometric information is ignored, resulting in consequent degradation of the technique as a means of identification. In many instances, SIM operation (rather than scanning of the full spectrum) is demanded both by the complexity of the mixture to be analysed and by the sensitivity required. In these instances, the aim must 0 1982 Elscvier Scientilic Publishing Company

The very rapid growth of mass spectrometry (MS) as a quantitative tool in the last 5-10 years has tended to promote a somewhat non-discriminatory approach, where its value in certain areas is overstated but where its merits in other demanding applications are not fully exploited. It is the purpose of this review to emphasize the important areas of application of quantitative MS, to evaluate recent refinements in technique and to project likely future developments. It is in the area of specificity that MS has the greatest advantage over other quantitative techniques. For many analyses of, for example, steroid hormones and drugs, the numbers of routine assays required is prohibitively high for the application of MS and cheaper, simpler methods (such as immunoassays or purely chromatographic procedures) with higher throughput are preferable. In many cases, routine assays may be usefully validated by comparison of relatively small volumes of data with those obtained by MS (though the concept of the ‘definitive’ method is questionable). Comparisons with routine techniques such as immunoassays suggest another important advantage of MS methods, namely that of versatility and consequent speed of method development. In the case of drug analyses, a functioning method employing gas chromatography-mass spectrometry (GC-MS) may often be developed in a matter of days or weeks, whereas development of an immunoassay, involving antigen synthesis and antibody raising, may take months of work. 0 I65-9936/82/0000-OOOO/WJ2.75

trends in analytical chemis&y, uol. I, no. 5,1982

be to maximize the information content of signals detected during selected ion monitoring. Put another way, the objective is to maximize specificity at high sensitivity. The extent to which new MS techniques render the twin aims of specificity and sensitivity compatible is discussed below.

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(C2H512NCH2CH2

Enhancement of analytical selectivity Several approaches have been adopted to achieve increased selectivity during SIM; they relate either to the means of production of ions or to their characterization. It is generally appreciated that high mass ions are more characteristic of an analyte than low mass fragment ions which have many possible origins. An ion incorporating the intact molecule is most characteristic; production of such ions may be favoured by the use of low energy electron impact ionization (affording M+’ ions) or by the use of a ‘soft’ technique such as chemical ionization (effecting, for example, protonation or other adduct ion formation). The latter is additionally valuable in that judicious choice of reagent gases provides the opportunity for selective ionization of certain components of complex mixtures. Thus, rather than achieve the essentially indiscriminate ionization of all volatile mixture components, as in the conventional electron impact (EI) technique, conditions may be adjusted to effect ionization only of chosen compound types.

II

concentration of 125 pg/ml, following a very simple work-up procedures. As an additional approach to sensitive and selective analyses, Dougherty3 has advocated the use of hydrocarbon mixtures with methylene chloride and/or oxygen as negative chemical ionization

Negative chemical ionization mass spectrometry The rapidly developing technique of negative chemical ionization mass spectrometry (CIMS) has been used to achieve analyses of high sensitivity and specificity. Major ion forming processes (reviewed by Dougherty”) include resonance or dissociative capture of low energy electrons and ion-molecule reactions. Detection of analytes with high erectron affinities may be uniquely sensitive. Thus, for example, Markey and co-workers4 have reported the detection of 5 X 10-14 g of the spirocyclic pentafluoropropionyl derivative of authentic melatonin (I) and have applied the procedure to its detection in blood plasma at the low picogram level. It is particularly noteworthy here that the high sensitivity of detection is attributable in part to the suitable choice of derivative; there is no doubt that selective derivatization procedures have a major part to play in the more widespread application of negative CIMS.

)1 dox50

I

The high selectivity of detection achievable in analyses of biological fluids and tissues has been illustrated by the studies of Garland and co-workers. Fig. 1, for example, illustrates the GC-negative CIMS/ SIM detection of flurazepam (II) in blood plasma at a

Fig. 1. CC-negative CIMSISIM analysis qfan aliquot (one etehth) of an extract of plasma containing 0.0125 nglml jlurazepam and 2.5 ng pH,v]jluracepam (internal standard). The arrows indicate the analyte and internal standard. Methane was the GC cartier gas and CI reagent gas. (Figure reproduced with permission from Miwa, B. J., Garland, W. A. and Blumenthal, P. (1981) Anal. Chem. 53, 796. Copyright 1981 American Chemical Society.)

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reagent gas; high selectivity in the detection of oxidizing agents and alkylating agents (both important in environmental applications) is observed whereas neutral lipids are virtually ‘transparent’.

Ionization of involatile compounds Exciting recent developments in the qualitative analysis of involatile compounds, with ionization achieved, for example, by fast atom bombardments, will undoubtedly be quickly followed by quantitative applications. In these instances, relined techniques of ion characterization will be particularly important.

High resolution selected ion monitoring

I

In most applications of quantitative MS, characterization of ions is based on the nominal mass-to-charge ratio. Significant improvements in selectivity may be achieved by increasing the MS resolution to detect only those ions of a chosen exact mass which correspond to a particular elemental composition7. This technique has now been used extensively in biochemical analyses and has recently gained prominence in environmental work with application to the determination of tetrachlorodibenzo-p-dioxin (TCDD)*. Clearly, operation at high MS resolution necessarily reduces signal intensity but in the analysis of complex mixtures, use& sensitivity my be increased by a greater reduction in the signal attributable to instrument background or to matrix constituents other than the analyte of interest.

I

I

Monitoring of ion fragmentations A further means of ion characterization is achieved by monitoring specific fragmentations of a chosen parent ion. In its simplest form, this approach involves the monitoring of metastable peaks which correspond to fragmentations occurring in the first field-free region of a double-focusing mass spectrometersYiO. The essential feature of the technique (which it shares with the more elaborate counterparts discussed below) is that the instrument conditions for signal detection are dictated by the mass-to-charge ratios of both parent and daughter ions, with a consequent increase in the specificity ofdetection. As with high resolution selected ion monitoring, the implicit sacrifice in absolute signal intensity is in many cases more than compensated by the reduction in interfering signals. This is dramatically illustrated in Fig. 2, which shows the GC-MS analysis of a plasma extract fraction, as the tertbutyldimethylsilyl (TBMS) derivative, with selected monitoring of ions of m/z 271 ([M-C.+Hs-H-

i.

J

I

2

6 A

1

,10

A

min.

1’ I

I

2

6

.

B

Fig. 2. GUMS of the tert-butyldimethylsilyl (TBDMS) dcrivativc of a j&ion obtained by simple gel chromatographic separation of a male blood plasma extract. A. Selected detection of ions m/z 271 formed in the ion source. B. Selected &tection of ions of ml2 271 f onned byfragmentation of metastable ions of ml< 347 in the jrst field-jee region of a double focusing mass spectrometer. The arrow indicates the peak corresponding to 5~ dihydrotestosterone TBDMS (see structuresIII); other peaks are attributable to isomeric androstanolones. (VG 70-70F mass spectrometer, m/Am 800; GC column 20 m x 0.33 mm glass coated with OV-I; temperature programmed analysis, 225260”, elmin.) (Figure reproduced by permission from Quantitative Mass Spectrometry in Life Sciences II, De Leenheer, R. R. et al. (eds), Elsevier, Amsterdam, 1978).

TBDMS ethers; (CHs)sSiOH] + for androstanolone III) formed in the ion source (Fig. 2A) or formed in the first field-free region of the double-focusing mass spectrometer by fragmentation of ions of m/z 347 (Fig.

2B).

mlz

m Iz 271

x7

m

The use of mass-analysed ion kinetic energy spectroscopy (MIKES)‘* provides a means of monitoring fragmentations of selected parent ions occurring in the second field-free region, between magnetic and electric sectors, of a reverse geometry double focusing mass spectrometer. The in,corporation of a collision cell promotes fragmentation. MIKES has been suggested as an alternative to GC-MS for qualitative and

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quantitative analysis of mixtures13. In many cases, however, such as the analysis of mixtures of isomers (Fig. 2), the value of techniques of selected fragmentation monitoring will lie in their ability to enhance the specificity of analyses based on tandem chromatography-mass spectrometry. Further refinements of this general approach in quantitative mass spectrometry may be expected, based on the use of multiple analyser instruments, where unit to high resolution of both parent and daughter ion beams is simultaneously achieved.

Conclusions Many developments in quantitative mass spectrometry have contributed to the utility of the technique. Advances in computerized data handling, for example, have encouraged the development of automated procedures and may enhance analytical precision. The more widespread availability of stable isotopically-labelled internal standards also contributes to improved precision and lessens the risk of systematic error. This review has concentrated on means of achieving enhanced specificity and sensitivity, features of quantitative mass spectrometry which particularly recommend its application to demanding problems in trace analysis. Such developments, of course, should be viewed as components of overall analytical strategy, and elegant techniques of instrumental analysis should not be used to disguise the inadequacies of poor procedures of sample preparation or sample introduction. Where due attention is paid to all aspects of analysis, however, recent developments in quantitative mass spectrometry open new areas of application and substantially improve the reliability of analytical data.

Reference

materials

References 1 Henneberg, D. (1959) Z. Anal. C&m. 170,369 2 Sweeley, C. C., Elliot, W. A., Fries, I. and Ryhage, R. (1966) Anal. Chem. 38, 1549 3 Dougherty, R. C. (1981) Anal. Gem. 53,625A 4 Markey, S. P., Lewy, A. J. and Colbum, R. W. in A. P. De Leenheer, Roncucci, R. R. and Van Peteghem, C. (eds) (1978) Quantitative Mass Spectrometry in Lift Sciences II, p. 17, Elsevier, Amsterdam 5 Miwa, B. J., Garland, W. A. and Blumenthal, P. (1981) Anal. them. 53, 793 6 Barber, M., Bordoli, R. S., Sedgewick, R. D. and Tyler, A. N. (198l)J. Chem. Sot., Chem. Commun. 325 7 Millington, D. S. (1975) J. Steroid Biochem. 6, 239 8 Hummel, R. A. and Shadoff, L. A. (1980) Anal. C&n. 52, 191 9 Gaskell, S. J. and Millington, D. S. (1978) Biomed. Mass Spectrom. 5,557 10 Gaskell, S. J., Finney, R. W. and Harper, M. E. (1979) Biomed. Mass Spcctrom. 6, 113 11 Gaskell, S. J. and Millington, D. S., in De Leenheer, A. P., Roncucci, R. R. and Van Peteghem, C. (eds) ( 1978) Quantitative Mass Spcctrometry in Life Sciznces II, p. 135, Elsevier, Amsterdam 12 Beynon, J. H., Cooks, R. G., Amy, J. W., Baitinger, W. E. and Ridley, T. Y. (1973) Anal. Chem. 45, 1023A 13 Kondrat, R. W. and Cooks, R. G. (1978) Anal. Chum. 50,81A Simon Gaskell obtained his undergraduate andgraduate degrees jiom tke University of Bristol, U.K. Following a post-doctoral fclowship at t/u University of Glasgow, he took up his present position in 1977 as head of the Mass Spectrometry Unit at the TenovusIn&u& for Cancer Research, Welsh National School of Medicine, Car&z CF4 4Xx, U.K.

for marine

trace analvsis _

_____~_

l

___

As we intensify our use of marine resourc&, accurate analysis of marine materials becomes kweaslrigJy important and reference materials are reqtiiied.

Roger Guevremont and W. D. Jamieson Halifax, Nova Scotia, Canada The establishment and implementation of regulations designed to control pollution ultimately requires accurate analytical data. Questions such as ‘how much of a certain toxic substance can safely be added to the marine environment?‘, must be answered in order to fix realistic safe limits for the disposal of toxic wastes. Are accurate chemical analytical methods available to measure the concentrations of such substances? Will 0 165.9936/82/0000-0/$02.75

available methods be used cqgsistently and conscientiously to ensure that Unsafe materiils are not unwittingly (or deliberately) dumped int6 the marine environment? To ensure these questions are adequately answered and as part of its role in fulfilling the International Convention on the Prevention of Marine Pollution by Dumping Wastes and Other Matter, the Canadian government established an Ocean Dumping Control Act and Regional Ocean Dumping Advisory Committee’**J. The regulations require that debris to be dumped at sea, most commonly the dredge spoils @ 1962 Elscvicr Scientific Publishing Company