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Reagent blanks It seems imperative to start any discussion on sample preparation with reagent blanks. In practically all applications relying upon solution nebulization of aqueous matrices, water is the most frequently used reagent. Prior to ICP-MS analysis digests of solid samples are usually diluted 100 fold with water, and thus the purity of the latter is of the utmost importance. Though ultra-pure water with guaranteed impurity contents at or below the ng l-1 level is commercially available, because of the challenges involved in avoiding contamination during transport and storage, as well as cost issues incurred when considering the volumes consumed in the average analytical laboratory, most laboratories rely upon in-house water purification systems. The current industry standard for the purification of tap water in analytical laboratories involves a combination of reverse osmosis and ion exchange processes. With deference to the name of the largest supplier of purification equipment, the resultant de-ionized water is often referred to as Milli-Q water.

To evaluate impurity levels in Milli-Q water (laboratory de-ionized water, purified first by reverse osmosis and then by four ion-exchanger cartridges connected in parallel), three stills (feed and receiving containers connected at 90 by elbows, all in Teflon, parts from Savillex Corp., Minnetonka, Minnesota, USA) were filled with approximately 1 l of water directly at the tapping point situated in a clean laboratory area with HEPA filtered incoming air. At the same time, an aliquot was collected into a Teflon bottle (reference sample). The stills were tightly closed, mounted on a heating device and water in the stills was evaporated at 80 C until only approximately 50 ml was left in the feed containers, thus providing a pre-concentration factor of approximately 20. This remaining water was acidified by addition of in-house distilled HNO3 (2% by volume or 0.28 M) and analysed by ICP-SFMS using an instrument reserved for clean reagents only. Blank subtraction was accomplished using acidified reference water. The following elements were present in pre concentrated water: Si (mean concentration from three aliquots was 1.2 g l-1);

B (200 ng l-1);

Zn (25 ng l-1);

P (17 ng l-1);

Al (11 ng l-1);

Fe (8 ng l-1);

Ni (4 ng l-1);

and Cu (2 ng l-1). The corresponding relative standard deviations for these mean values were below 20%. For Na, K, Ca and Mg (mean concentrations in the low ng l-1 range), standard deviations were above 30% probably reflecting variable contamination from stills and sample handling. Concentrations below 1 ng l-1 were found for the rest of the approximately 60 analytes studied.

This experiment demonstrates that, except for Si, B and Zn, all other elements are present in fresh Milli-Q water at sub-ng l-1 concentrations (actually below 50 pg l-1 for the majority of tested analytes), i.e., at levels sufficiently low for almost any application. Occasionally, concentrations of some elements (mainly Si and B, but also Al, Ba and U) may increase in de-ionized water either as result of sudden changes in the pressure of the feeding water or when approaching the loading capacity of the ion-exchangers. Sub-boiling distillation in Teflon stills provides a simple and cost efficient means to purify Milli-Q water further and to ensure good water quality independent of the life span of the ion-exchanger cartridges, though slight increases in the concentrations of some common analytes (i.e., Na) can be expected due to extra handling of the water. This water is herein referred to as distilled de-ionized water (DDIW). Typical concentrations of a few selected elements measured in laboratory water after various purification steps (reverse osmosis ELIX water, Milli-Q and DDIW), as well as commercially available ultra-pure water are summarized in Table 1. As these values are not blank corrected, they all include the instrument blank component (see below), but because samples were analysed in the same analytical session, this contribution is expected to be the same regardless of water source. For the majority of elements, differences in water purity between different sources can be detected only at low or sub-ng l-1 concentrations. As a rule, both Milli-Q and DDIW are of similar purity to commercial ultra-pure water. It should be noted that the Milli-Q purification unit used for these experiments is almost a decade old and even better water quality can be expected with modern systems incorporating additional filtering or polishing stages.

Table 1. Concentrations of selected elements in ultra-pure water from different sources 1% HNO3 in 1% HNO3 in Milli- 1% HNO3 in 1% HNO3 in Element Unit ELIX water Q water DDIW water Fisher water g l- S 1.21 (0.02) 1.22 (0.02) 0.78 (0.02) 1.31 (0.03) ng l- Na 320 (20) 9 (2) 18 (2) 21 (2) ng l- Al 25 (6) 11 (1) 3.5 (0.7) 4.4 (0.9) ng l- Mg 16 (3) 2.0 (0.2) 1.3 (0.3) 0.52 (0.03) ng l- Fe 11 (2) 3.6 (0.7) 1.2 (0.1) 1.5 (0.2) ng l- Ba 10 (1) 1.2 (0.2) 0.025 (0.003) 0.031 (0.005) ng l- Cu 5.7 (0.7) 3.8 (0.3) 1.7 (0.2) 1.7 (0.2) ng l- Mo 1.3 (0.1) 0.65 (0.03) 0.11 (0.03) 0.64 (0.04) pg l- W 600 (90) 72 (12) 23 (2) 260 (20) pg l- Pb 350 (30) 110 (10) 31 (15) 68 (8) pg l- Sb 110 (10) 28 (3) 10 (1) 150 (10) pg l- U 3.6 (0.5) 2.8 (0.5) 2.3 (0.4) 12 (2) As with water, frequently used acids (HNO3 and HCl) can be easily purified in-house from low-grade (i.e., PA) feed using sub-boiling distillation in quartz or Teflon stills. When distillation is done continuously under optimized conditions, the purity of the resulting acid is comparable to that of commercially available supra pure grades and for the majority of elements the contribution from the acid in a 10% by volume solution of HNO3 (1.4 M) in Milli-Q water is negligible. When only limited volumes of acids are necessary (typically HF or HClO4), purchasing commercial ultra-pure acids can prove to be a more cost effective alternative compared to setting up in-house purification.

However, one should be prepared for possible variations in purity between different batches. For example, we have observed that the level of S in some batches of ultra-pure HF may be orders of magnitude higher than PA grade supplies. At the ng l-1 level, higher levels of elements in some batches are not uncommon in acids and other laboratory chemicals, e.g., Sn or Ti in H2O2.

All the previous discussion on laboratory reagent purity was related to freshly produced, or, in the case of commercial products, newly opened chemicals. During the preparation of solutions, levels of impurities in water and acids can increase significantly because of leaching from storage containers, contact contamination from pipette tips or dispensers, or accumulated contamination from the laboratory environment while containers are open. Data presented in Table 2 demonstrate changes in concentrations of 24 elements in Milli-Q water during handling in the laboratory. Five sub-samples of water were tapped into thoroughly acid cleaned 2 l HDPE bottles. One bottle was set aside to be used as reference sample, while the remaining four were used for routine preparation at different locations in the laboratory. At the end of the working shift, all water samples were acidified to 2% by volume HNO3 (0.28 M) and analysed by ICP-SFMS. Increased concentrations were found for many elements (Table 2) with some waters accumulating up to 100-fold higher levels of selected analytes compared to the original contents. This is in spite of the fact that all handling of water containers was performed in a clean laboratory by with personnel wearing clean room clothing, hair nets and gloves, following routines to limit handling contamination, including rinsing of all pipette tips in a sequence of supra pure HNO3 then Milli-Q water prior to use.

Table 2. Concentrations of selected elements in ultra-pure water before and after use in laboratory Milli-Q after 8 h, Milli-Q after 8 h, Milli-Q on tap, Milli-Q on tap, Element Element range (n=4) range (n=4) ng l-1 ng l- ng l-1 ng l- Al 11 40-120 Mg 2 9- Ag 0.9 0.9-1.4 Mn 0.6 0.7-2. Be 0.02 0.03-0.3 Na 10 100- Bi 0.01 0.02-0.5 Ni 4 4- Ca 20 100-700 Pb 0.06 0.2-1. Cd 0.02 0.03-0.2 Rb 0.1 0.2-1. Co 0.08 0.07-0.2 Sb 0.01 0.02-0. Cr 0.3 0.4-2 Sr 0.2 0.5-1. Cu 2 2-12 Th 0.004 0.004-0. Fe 4 5-60 Ti 0.2 0.3- Ga 0.01 0.03-0.08 V 0.01 0.02-1. K 20 100-1300 Zn 4 11- It is interesting to note that concentrations of such common elements as Fe and Zn have increased 15 times even in the most contaminated bottles. At the same time, significantly higher maximum increases in the 30- to 50-fold range were found for the relatively environmentally uncommon elements, Bi and Sb. The latter elements most probably originated from the use of make-up, where they are used at high concentrations either as additives for inhibiting bacterial grow in lipid-rich formulations or as a bulk component where stibnite constitutes the black pigment in mascara. Though the wearing of decorative cosmetics is prohibited in the laboratory, minute amounts of make-up may still contaminate a clean environment as residue on skin, under nails, etc.

It should also be mentioned at this juncture that settling dust is obviously of concern at all stages where the sample is exposed to the laboratory atmosphere, as would be the case during the course of these experiments. Measurements performed in this laboratory showed that the dust sedimentation rate on the surface of an instrument was reduced from 3000 to 20 pg cm-2 h-1 by the introduction of a HEPA filtered air supply. Therefore the increases in concentrations evident in the results summarized in Table 2 are too great to be explained solely by contamination by settling dust, at least for the elements Al, Ca, Fe, K, Mg, Na and Zn.

Another source increasing the concentrations of uncommon elements in laboratory air is the dilution of stock 1000 mg l-1 standards by pipetting, a process that can result in the formation of tiny aerosols contaminating the atmosphere and surrounding preparation area surfaces. As a result, the accumulation of ultra-trace analytes (e.g., Ir, Re, Tl) can be detected in reagents and standards handled in the vicinity of the standard preparation area. The reservation of area dedicated to the dilution of trace element standards with concentrations above10 mg l-1 is therefore recommended in order to limit the risks for such contamination.

Laboratory ware Sample containers are potential sources of contamination as well. This contribution can be minimized using sample bottles with large volume-to-surface ratios (1 or 2 l volume) made of virgin Teflon and thoroughly acid washed prior to use. One of the important advantages of Teflon is its ability to withstand the high temperatures and concentrated acids necessary for efficient cleaning. Though this approach is widely used for the analysis of ultra-pure waters and other reagents used in semiconductor applications, it can not be recommended for all samples because of limited throughput (i.e., cleaning) and cost considerations. As the majority of routine applications rely on the use of autosamplers, sample tubes with volumes in the range 115 ml are most frequently used. Though such autosampler tubes are available in Teflon, disposable tubes made of polystyrene and polypropylene are much more widespread. In the selection of a specific tube variety suitable for a given application it is therefore important to consider the amounts of target analytes that can be leached into the sample matrix during preparation, storage and analysis. The amounts will depend on the materials comprising the tube/stopper/cap, the washing procedure (if any), the strength of the acid (or acid mixture) in the sample, the duration of contact, and the temperature during leaching.

Table 3 summarizes leaching tests for 15-ml polypropylene tubes. This includes 12 h leaching of unwashed tubes (5%- and 1%-by volume HNO3), tubes washed in a metal-free laboratory dishwasher using a mixture of hot HNO3/HCl (PA grade) followed by 20% by volume HNO3, and washed tubes that have been filled with 5% supra pure HNO3 for storage and rinsed with Milli-Q water before use. The efficiency of this washing procedure is evident from the significant decreases in the leachable concentrations of many analytes, though keeping washed tubes filled with weak acid and applying an additional rinse helps reduce contamination from tubes even further. From our experience in testing a variety of disposable polystyrene and polypropylene autosampler tubes available on the market, the following general observations have been made:

All tubes are sufficiently clean as supplied to be used for geological and regulation focused environmental applications.

None of the tube types is clean enough to be used as supplied for applications requiring measurements of common elements at sub-g l-1 concentrations.

Even pre-washed tubes can release significant quantities of some elements (Al, Si, Zn, Sn, Ti, Cd) in contact with relatively concentrated acids (i.e., 5% by volume HNO3).

This effect accelerates at higher temperatures.

Table 3. Effect of pre-treatment on leachable (12 hours at room temperature) concentrations from polystyrene 15 ml autosampler tubes As supplied, 5% As supplied, 1% Washed, Washed/rinsed, Element Unit HNO3 HNO3 1% HNO3 1% HNO (SD), n=12 (SD), n=12 (SD), n=12 (SD), n= g l- Ca 5.4 (2.9) 2.7 (0.6) 0.42 (0.41) 0.030 (0.029) g l- Na 3.8 (1.9) 0.65 (0.37) 0.28 (0.19) 0.041 (0.034) g l- Zn 1.5 (0.7) 0.66 (0.07) 0.19 (0.04) 0.004 (0.003) g l- K 1.4 (1.2) 0.41 (0.22) 0.069 (0.048) 0.018 (0.019) g l- P 0.75 (0.12) 0.49 (0.04) 0.030 (0.019) 0.005 (0.002) g l- Al 0.75 (0.24) 0.45 (0.21) 0.032 (0.020) 0.005 (0.005) g l- Fe 0.67 (0.39) 0.19 (0.14) 0.090 (0.050) 0.016 (0.009) g l- Mg 0.44 (0.31) 0.14 (0.04) 0.049 (0.038) 0.007 (0.010) ng l- Ti 130 (30) 74 (5) 4 (2) 0.7 (0.5) ng l- Cu 65 (50) 41 (32) 4 (5) 2 (1) ng l- Ba 56 (42) 41 (29) 1.2 (0.6) 0.3 (0.3) ng l- Pb 36 (24) 14 (12) 0.3 (0.4) 0.1 (0.1) ng l- Cd 12 (16) 4.1 (1.9) 0.04 (0.03) 0.01 (0.01) Handling contamination After considering some of the factors potentially limiting the detection capabilities, it is useful to evaluate the lowest concentration in the simple matrix at which reliable (accurate and reproducible) measurements can be performed. The following experiments were conducted for this purpose: by gradual dilution of 10 mg l-1 multi-element standards (Perkin-Elmer), a duplicate set of spiked solutions (concentration range 0-100 ng l-1) were prepared in 1%- and 5%-by volume HNO matrices. Acid-washed polypropylene tubes, DDIW and distilled acid were used in the preparation of these solutions following general procedures used for the analysis of pure industrial and natural waters in the laboratory. The concentrations of approximately 70 elements were determined in spiked solutions by an ICP-SFMS instrument reserved for the analysis of pure reagents (negligible long-term memory effects) using internal standardization (In added at 2 g l-1 to all solutions) and external calibration (at 1 g l-1). Duplicate samples were analysed in two separate sequences, one for each acid strength, with spiked solutions arranged in a random order.

The limit of quantification (LOQ) is defined as the concentration corresponding to 10 SD for blanks measured under repeatability conditions [16]. While frequently perceived as a useful measure of performance in analytical studies, the LOQ calculated in this fashion can prove to be overly optimistic. Consequently, more modern guidelines [17] generally include some requirement for validation of the LOQ determined as above. Here the lower limit of reporting (LLOQ) for each analyte was set at the lowest spike concentration that could be measured with the following criteria fulfilled: average found concentration (two separate preparations/analyses) within 20% of the theoretical value;

relative standard deviation for two replicates below 20%;

all spike solutions at higher concentrations satisfy the first two requirements as well (see Table 4). Though probably not the most familiar means of assessing the detection capability in the analytical community this approach serves to validate the results in the fashion specified in reference [17]. It also provides important information on the efficacy of ICP-SFMS for determinations at low concentrations using routine measurement conditions (hot plasma, samples prepared in disposable autosampler tubes, solution delivered to nebulizer by peristaltic pump, analyses performed in truly multi-element mode with an effective measurement time of 1 s per isotope, etc.).

Table 4. Detection capabilities of ICP-SFMS in diluted HNO LLOQ, ng l-1 1% HNO3 (0.14 M) 5% HNO3 (0.7 M) 0.05 Ag, Cs, Eu, Ir, La, Nb, Nd, Pt, Re, Rh, Ru, Cs, Eu, Ir, La, Nb, Nd, Pt, Re, Rh, Ru, Sb, Sm, Tb, Sb, Sm, Tb, Tl, Tm, U, W Tl, Tm, U, W 0.1 Au, Ce, Dy, Er, Gd, Ho, Lu, Pd, Pr, Ta, Y, Yb Ag, Au, Ce, Dy, Er, Gd, Hf, Ho, Lu, Pd, Pr, Y, Yb 0.25 Cd, Co, Ga, Rb, Ru, V Cd, Co, Ga, Rb, Ru, Th, V 0.5 As, Be, Cr, Ge, Mo, Pb As, Be, Bi, Ge, Mo, Pb, Zr 1 Ba, Cu, Mn, Sc, Sn, Sr, Te, Hg Ba, Cr, Cu, Mn, Sc, Sn, Sr, Ta, Te, Hg 5 Li, Mg, Ni, Ti, Zn Li 20 Al, B, Ca, Fe, K, P, Se Al, B, Ca, Fe, K, Mg, Ni, Se, Ti, Zn 50 Bi, Na Na, P 100 Hf, Th, Zr 100 Si, S, Br Si, S, Br LLOQs at or below the 1 ng l-1 level are possible to achieve for more than half of the elements tested. It is interesting to note that in spite of low blanks, it was impossible to obtain reliable results for Bi, Th, Hf and Zr using solutions prepared in 1% HNO3, as recoveries of spiked concentrations were too low. The effect is demonstrated in Table 5, where measured concentrations for selected elements in blanks and samples spiked at 1, 5, 20 and 50 ng l-1 are presented. It seems that low recoveries for some elements present in solutions with low acid strength are caused by losses of analytes on the tubing. Those trapped quantities can be eluted by either increasing the HNO3 concentration in the rinse solution to 5% by volume (Bi) or by using traces of HF (Th, Hf, and Zr). This phenomenon is much less pronounced for spike solutions prepared in 5% by volume HNO3. These findings have two important implications. Firstly, the accurate determination of these elements in such matrices as surface waters, melted snow and pure industrial solutions would require sample acidification at or higher than 5% by volume HNO3. Secondly, elements deposited on tubing surfaces can result in false signals in subsequent solutions, particularly in cases where variable acid matrices are analysed, e.g., when some of the samples have been digested using mixtures containing HF.

Table 5. Spike recovery at ng l-1 range and memory effects in introduction system Measured Re Measured U Measured Bi Measured Th Measured Zr Position concentration, concentration, concentration, concentration, concentration, ng l-1 ng l-1 ng l-1 ng l-1 ng l- Blank 1 (1% HNO3) 0.001 0.007 0.002 0.001 0. Spike 1 ng l-1 1.07 1.00 0.34 0.21 0. Spike 5 ng l-1 5.09 5.09 1.31 0.42 0. Spike 20 ng l-1 20.5 21.1 5.79 1.55 3. Spike 50 ng l-1 50.3 48.8 45.2 19.1 20. Standard 100 ng l-1 100 100 100 100 Blank 2 (1% HNO3) 0.11 0.14 0.65 0.18 1. Blank 3 (1% HNO3) 0.038 0.036 0.15 0.07 0. Blank 4 (1% HNO3) 0.005 0.002 0.06 0.05 0. Blank 5 (5% HNO3) 0.005 0.051 7.9 5.9 Blank 6 (5% HNO3) 0.004 0.031 1.7 2.7 Blank 0.002 0.023 0.62 13 (5% HNO3+0.05% HF) Analysis of clean water samples as described above can be considered one of the favourite (and simplest) ICP-MS applications due to negligible matrix effects, predictable spectral interferences and limited handling contamination. For matrices requiring extensive preparation, the latter component often becomes the major obstacle defying the attainment of equivalent LLOQs to those that can be achieved for water samples. Let us consider the analysis of whole blood after digestion with HNO3 [12]. Briefly, 1 ml of blood is digested with 1 ml HNO3 in a closed Teflon vessel using a laboratory microwave oven. The digest is diluted to 10 ml with Milli-Q and analysed by ICP-SFMS. All plastic ware used in the analysis is disposable except for the digestion vessels themselves, which are cleaned between runs in a sequence with ethanol and 50% by volume HNO3, followed by rinsing thoroughly with Milli-Q water.

Table 6 summarizes figures of merit for this procedure assessed from analysis of approximately 2200 whole blood samples taken for routine health evaluation from Scandinavian donors. Method LOQs for this particular study were evaluated as average blank concentrations (one preparation blank per 20 patient samples was prepared using 1 ml Milli-Q water instead of blood) plus 10 SD of these preparation blanks (n 100). While this definition of the LOQ is also unconventional (c.f. Keith et al. [16]), it accounts for day-to-day variations in every link in the analytical chain and thus faithfully reflects the capabilities of a method in routine use. Method reproducibility was assessed using replicate preparation and analysis of randomly selected samples (relative standard deviation, n = 50). Mean/median concentrations were calculated using the entire data set without exclusion of outliers. For Cd, Hg, Tl and U, LOQs for the whole blood method are only a few times poorer than for clean water analysis (c.f. Table 4, taking into consideration the 10 fold dilution for the blood samples) reflecting more extensive sample handling and minor variations in long-term instrument performance (data were acquired from 20 separate measurement sessions). Since neither a shielded torch nor an X-skimmer cone are employed in the introduction system of the ICP-SFMS instrument dedicated to body fluid analyses, lower sensitivities (by factors of 10-15, see following section) may add to higher LOQs as well. The median blood U concentration (15 ng l-1) is within the range of blank levels leaching from common tubes used for blood collection (5-20 ng l-1, [18]), suggesting that meaningful determination of this element in blood collected using these tubes is impossible. Depending on the type of collection tubes this contribution can be significant for V, Cr, Co, Ni, Li and Tl.

Table 6. Detection capabilities of ICP-SFMS for blood analysis LOQ, g l-1 Mean/Median (n2000), g l- Element Mean RSD for duplicate samples (n=25), % Al 10 35 19/ Cd 0.014 8 0.38/0. Co 0.1 36 0.12/0. Cr 0.4 42 0.84/0. Cu 7 4 810/ Hg 0.07 7 2.2/1. Li 0.9 9 11/ Mn 0.6 7 7.9/7. Mo 0.1 4 1.2/1. Ni 0.6 25 1.2/0. Pb 0.3 4 19/ Sn 0.2 29 0.23/0. Tl 0.002 12 0.026/0. U 0.003 15 0.023/0. V 0.05 13 0.15/0. Zn 12 4 6200/ For elements such as Al, Zn and Cu, LOQs for blood are poorer than those for water by almost three orders of magnitude. Handling contamination is much more severe for these common elements and also memory effects from blood samples may contribute to blank levels for the last two analytes. Owing to the higher LOQs, it was impossible to achieve good reproducibility for Al, Co, Cr, Ni and Sn in blood samples from the occupationally unexposed population using the routine method (Table 6). On the other hand, high LOQs can be tolerated for Cu, Mn, Pb and Zn as typical concentrations in whole blood are at least 10 times higher.

Instrumental analysis To cope with the demands for reliable ultra-trace analyses, a number of technical developments aimed at improving instrumental sensitivity have been implemented. For example, the typical sensitivity offered by a first generation ICP-SFMS instrument equipped with standard introduction system for mid-mass isotopes of abundance close to 100% (Rh, In or Cs) is in the range (80-120) Mcounts s-1 per mg l-1 with a sample consumption of (0.51.0) ml min-1. Improving the skimmer cone design (high performance skimmer) augmented this value to 200 Mcounts s-1 per mg l-1, decreasing the plasma potential using a shielded torch gave sensitivities in the range (1-2) Gcounts s-1 per mg l-1, and combined with the X-cone skimmer design up to 5 Gcounts s-1 per mg l can be achieved. By using the latest versions of the high efficiency nebulizer systems (Aridus or Apex), one can reach (2030) Gcounts s-1 per mg l-1 with a sample uptake of only 0.05 ml min-1.

Even higher sensitivities can be obtained for selected analytes using the generation of gaseous species (cold vapour, hydrides or oxides) from measurement solutions, though these gains come at the expense of sample volume and multi-elemental capabilities.

Improvements in ion-path designs and in the quality of electronic components have resulted in the instrumental background, defined as the signal monitored at a mass free from natural isotopes (e.g., 220 amu), decreasing from (50100) counts s-1 to (12) counts s-1 (or 0.2 counts s-1 for sector instruments). Theoretically, the detection capability of modern instrumentation should easily reach the sub-ng l-1 or even sub- pg l-1 region. In reality, however, several additional instrumental factors complicate the possibility to reach such low levels in real samples.

Firstly, in order to use the widespread solution nebulization introduction system, solid matrices have to be converted into solutions by means of dissolution, digestion or sintering/fusion, effectively resulting in dilution factors that directly affect LOQs. Even for liquid (aqueous) samples with concentrated matrices (brackish and seawaters, body fluids, alcoholic and non-alcoholic beverages, etc.) dilution is required in order to control matrix effects. The degree of dilution depends upon the instrumentation used and the configuration of introduction system;

usually the ones providing the highest sensitivities have been most prone to matrix effects. In some applications, matrix separation/analyte pre-concentration can be successfully used to eliminate the need for sample dilution. Apart from involving additional sample handling, this approach limits multi-element capabilities as it is often applicable to only a limited number of analytes [1921].

Secondly, detection capabilities can be affected by spectral interferences originating from plasma gases, solvent and sample matrix. The importance of proper corrections for accurate measurements at low concentrations can hardly be overemphasized, and there are a number of useful reports on this topic [2224]. Among the approaches developed to deal with spectral interferences are the selection of less affected isotopes, the chemical separation of interfering elements, plasma parameter optimization, the use of mixed or alternative gas plasmas, reaction/collision cells and high resolution. Unfortunately, none of these approaches is capable of eliminating all potential spectral interferences, and instrumental performance (versatility, measurement time and sensitivity) can often be affected. For example, significant reductions of sensitivity using collision cells (for low mass analytes) or sector instruments in high resolution mode (for all analytes) may affect instrumental detection capabilities.

Thirdly, measured analyte signals may contain contributions from instrumental background and contamination during sample preparation. According to experience gained from applications of ICP-SFMS to a great variety of sample types, instrumental sensitivity is seldom the decisive factor as in the majority of cases the detection capabilities are blank limited. Aiming at world record sensitivity in excess of 1010 counts s-1 per mg l-1 is unnecessary given that even instrumental (not to mention preparation) blanks can be in the (1001000) counts s-1 range for many common analytes of broad interest. Given that the instrumental configuration and measurement parameters providing the highest sensitivity are usually sub-optimal with regards to tolerance to matrix effects, will likely incur increased costs for the sample introduction system, and shorten the useful lifetime of some instrumental parts (e.g., the secondary electron multiplier or the slits), efforts focused on improving blanks are often preferable and ultimately more cost effective than gains in sensitivity.

Concluding remarks A plethora of factors have to be carefully considered to perform accurate and reproducible analyses at the ultra trace levels matching the performance offered by modern analytical instrumentation. Providing a detailed recipe that would be directly applicable to every analytical task is, of course, impossible. As for clean-sampling techniques [25], controlling unwanted contributions should be viewed as a flexible philosophy requiring chemical and analytical understanding by the practitioner, as well as a healthy dose of common sense. However, some general hints and starting points for method development for ultra trace analysis by ICP-MS can be suggested.

Acquire as much relevant information on the matrix and expected concentration levels of both analytes and potentially interfering elements in the samples studied as possible. The targeted purity levels should be fit for purpose to be cost-efficient.

While developing sample preparation/analysis protocols, limit the number of steps and general manipulations of the sample/sample digest, especially those open to the laboratory environment. The less laboratory ware (homogenizing equipment, vessels, vials, tubes, filters/filter holders, electrodes, pipette tips, magnet stirrers, etc.) that comes in contact with samples the better the chances to limit contamination.

Make sure that available reagents are of sufficient purity, instead of limiting volumes used. High reagent/sample ratios usually improve analyte recovery and it is often advantageous to use relatively high acid concentrations at the measurement stage. Be aware though that the purity of water and acid aliquots used for sample preparation may deteriorate rapidly while in use.

Pay proper attention to maintaining a clean laboratory environment. Instead of creating confined clean room zones, focus on isolating potentially severe contamination sources such as crushing, grinding, sieving, cutting and ashing operations, as well as handling highly concentrated standard solutions. It is advisable to ban the wearing of cosmetics and introducing changes of clothes/shoes before entering laboratory areas. With a carefully laid out and managed laboratory, sub-ng l-1 LOQs can be achieved routinely without needing to implement a Class-10 or even a Class-100 environment.

Develop adequate cleaning routines for disposable laboratory ware. For some common elements though, extensive washing procedures may not be effective because of contamination introduced during handling.

Make a thorough evaluation of potential spectral interferences, taking into account even seemingly unlikely species (including multiply charged, truly polyatomic ions) beyond the standard interference table offerings available from instrument producers. The lower the concentration of analyte, the higher the probability that minor, unusual interferences will adversely affect the accuracy. Select a suitable strategy to correct for, or preferably eliminate them depending on the severity of the interferences and the instrumentation available. Whenever possible, measure several isotopes per element to verify the efficiency of corrections.

Be prepared for unexpected sources of contamination in the laboratory. Test all detergents, hand wash formulations, lotions, etc., for potential impurities. Remember that metal free is not equivalent to sufficiently pure for all applications, e.g., a metal free dispenser may actually introduce measurable quantities of PGEs to dispensed solutions.

Periodically evaluate typical instrumental blanks for each individual instrument. As far as is practically feasible, try to implement a dedicated introduction system, or ideally, a dedicated instrument approach. Account for memory effects in introduction systems, and optimize the calibration strategy with regards to the concentrations of analytes in standards and their placement in the measurement sequence, etc.

A realistic assessment of the detection capabilities of the analytical method requires that a set of method blanks are analysed randomly in a sequence with authentic samples. For the preparation of such preparation blanks, make sure that all manipulations with real samples are replicated as closely as possible. Remember that, for analytes potentially affected by spectral interferences from matrix elements, LOQs derived from preparation blanks might nevertheless be completely irrelevant.

In order to verify measurement accuracy at ultra low concentrations, the development of new reference materials certified for as many trace elements as possible is necessary [26]. Until such materials become available the publication of surplus data obtained for non-certified elements in commercially available reference materials will aid performance evaluation in the mean time [27]. Where available, inter laboratory exercises, round robins and performance evaluation tests remain the most valuable option [28].

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Assessed 28 July 26. Krachler, M., Heisel, C., Kretzer, J.P., 2009. Validation of ultratrace analysis of Co, Cr, Mo and Ni in whole blood,serum and urine using ICP-SMS. Journal of Analytical Atomic Spectrometry, 24, 605- 27. Rodushkin I, Engstrm E, Srlin S, Baxter DC (2008) Levels of inorganic constituents in raw nuts and seeds on the Swedish market. Sci Tot Environ 392:290- 28. Rodushkin I, Engstrm E, Stenberg A, Baxter DC (2004) Determination of low-abundance elements at ultra-trace levels in urine and serum by inductively coupled plasma-sector field mass spectrometry. Anal Bioanal Chem 380:247- ROLE OF MASS SPECTROMETRY IN THE DEVELOPMENT OF ANTICANCER METALLODRUGS Svetlana S. Aleksenko1 and Andrei R. Timerbaev Institute of Chemistry, Saratov State University, Astrakhanskaya Str. 83, 410012, Saratov, Russia Vernadsky Institute of Geochemistry and Analytical Chemistry, Russian Academy of Sciences, Kosygin St. 19, 119991, Moscow, Russia INTRODUCTION There is currently a significant interest in developing anticancer metallodrugs, as evidenced by numerous conferences, original and review articles, books and book chapters. Amongst the approved metal-based drugs, the Pt(II) complexes have gained most of celebrity because of their well-demonstrated antitumor properties. No surprise, therefore, is that their total sales are in excess of two billion dollars per annum. Alongside, several compounds of ruthenium(III) and gallium(III), exhibiting in vivo antitumor activity, progressed to commercial development.

In the human body, a metal-based drug is subject to a variety of metabolic processes that would change considerably the intact drugs composition and result in a complex metal-speciation pattern. Particularly, ligand-exchange reactions will be frequent in biological media, with both low and high-molecular-weight biomolecules capable to substitute the leaving ligands of the parent drug. In this regard, a substantial part of metabolism inherent to metal-based drugs (in view of their predominately intravenous administration), are reactions with plasma proteins that contribute in a varying degree to the fate of the administered drug. Since metabolic transformations are likely to occur before reaching the target, most metallodrugs are considered as pro-drugs. To underscore, the different biological compartments of interest, e.g., whole blood, plasma and its ultrafiltrate (pUF), or excreta, such as urine or feces, comprise a great variety of inorganic, organic, bio-, and mixed-ligand forms of a given metal, whose abundances (i.e. relative concentration levels) might differ to a large extent. For instance, up to 17 platinum-containing metabolites of oxaliplatin have been observed yet before drug entering the cancer cell as based on pUF analyses from cancer patients (Graham MA et al. Clin. Cancer Res. 6 (2000) 1205) There are several reasons why one needs to monitor the metallodrugs and their metabolites in biological samples. The first objective is systematic pharmacokinetic studies essential for the successful development and optimal use of a drug in chemotherapeutic treatment. Drug pharmacokinetic characteristics, such as the maximal drug concentration, free plasma concentration, terminal half-life, plasma clearance, etc., are derived directly from the analytical data obtained by analyzing plasma and pUF samples at different dose levels. Similarly, drug clearance, which is in the most cases identical with renal clearance (as renal excretion is the major pathway by which metal anticancer agents are excreted), is determined by monitoring drugs urinal levels. Another motive stems from toxic side effects that limit clinical treatments with certain metal-based chemotherapeutics, in the first place, cisplatin. Moreover, when overdosed, this drug may be carcinogenic and genotoxic in mammalian cells and this harmful feature appears to be of a special concern due to the risk of inducing secondary malignancies. In this context, changes in concentrations of metallodrugs after administration play an important role and require accurate measurements. Analyses of tumor, tissues, and organs samples and cell compartments from patients are answerable for the issues of drug uptake, accumulation, distribution, and long-term retention and also shed light on the drug mechanism of action. Last but not least for drug discovery, metabolism studies greatly rely on information concerning metabolite profiling. The latter implies separation, identification, and quantification of the major metabolites resulted from the biotransformation of an investigational compound.

Mass spectrometry (MS) is undoubtedly a premier technique in the field under consideration. The MS methods used for determination of metallodrugs, their metabolites and studying metallodrug interaction with biomolecules can be divided into two main groups:

(i) direct (or non-specific) methods, such as inductively coupled plasma or electrospray ionization MS (ICP-MS ans ESI-MS, respectively), that enable measurement of (only) total metal concentration in a given biosample, as an entity, or in its compartments after fractionation;

(ii) separation-based (or specific) methods in which the same MS techniques are combined with a separation technique (high-performance liquid chromatography (HPLC), capillary electrophoresis (CE), etc.). This allows different metal-containing species to be separated prior to MS detection. Separation-based methods can thus provide information on the identity and concentration of metallodrug metabolites and adducts with biomolecules present in the biosample.

The advantages and limitations of different MS techniques, introduced in Table 1, are critically scrutinized in the main part of this lecture using examples taken from recent literature and authors research.

Table 1 Comparison of different MS techniques used for developing anticancer metallodrugs Method Application domains Advantages Requirements Disadvantages ICP-MS Independent Highest elemental Separation step No structural quantification of sensitivity and occasionally information unbound drug and drug Element-specific and sample Spectral interferences adducts at multi-element pretreatment by and matrix effects in physiologically information microwave real-world analysis relevant concentration Moderate sample digestion Expensive (including analysis of amount requirement instrumentation and clinical samples) Availability of high maintenance Direct determination of advanced detection costs degree of protein technologies Adsorption effects in metalation (reaction/collision case of ultrafiltration Pharmacokinetic cell, high-resolution separation investigations mass analyzers, etc.) ESI-MS Direct confirmation of Precise molecular Special Insufficient sensitivity the formation of drug mass information requirements for (with conventional adducts Non-destructive volume, instrumentation) Accurate structural analysis (the softest concentration, Limited applicability characterization ionization method) and composition to clinical samples (including weak Sample consumption of the sample Compatibility adducts) (nano-ESI-MS) solution, constrains for large Kinetic measurements depending on the proteins type of mass analyzer CE-ICP- Kinetic measurements Minimum effect of Modification of No information on MS Determination of separation system on standard adduct molecular binding constants and chemical integrity of separation setup identity stoichiometry analytes Need of Adsorption effects on Quantification of Species and element experienced capillary walls and in proteindrug adducts in selectivity personal the interface biomatrices and Multi-elemental Insufficient sensitivity samples (ex vivo detection capability due to make-up analysis) Minute sample dilution effect requirement Expensive detection Short analysts times unit HPLC- Kinetic measurements Fairly high sensitivity Use of Excessive cost of the ICP-MS Determination of Species and element collision/reaction instrumentation and binding constants and selectivity cells to conquer maintenance costs stoichiometry Multi-elemental matrix/spectral Occasional Quantification of detection capability interferences incompatibility of proteindrug adducts at Estimation of the Additional detection performance clinically relevant molecular mass protein digestion and analyte integrity concentrations distribution of step for structural with mobile phase and Metal-peptide mapping adducts (in SEC (mapping) studies matrix compositions Monitoring of metal- mode) Supplementary Low recovery of exchange processes Different use of ESI-MS analytes (because of (complementary) coupling for sorption onto the separation modes adduct structural stationary phase) Adequate stability of characterization Need of large sample analytes in the case of volumes in case of SEC analytical columns Robustness and Rather long analysis easiness to maintain time NON-SEPARATION MASS-SPECTROMETRIC METHODS Elemental Mass Spectrometry During the last two decades, ICP-MS has become a well established technique applied on a routine basis for the determination of trace elements in biological samples. Application of ICP-MS also extends to the field of metallodrug analysis where the method is most commonly used for quantification of the total metal content in samples obtained from patients who received chemotherapeutic treatment. The major points of excellence of ICP-MS are extremely low, unrivaled limits of detection (LODs) and independence of the detector response of the molecular structure of a given element. Thus, not only can metal-containing drugs be detected based on the signal of the metal atom but also quantified against an elemental standard. This also makes ICP-MS the technique of preferential choice for metabolite profiling, as the metal-containing metabolites can be determined without the need for authentic standards due to the structure independent response.

Nowadays, ICP-MS analyses are dominated by platinum-based drugs, with only few papers encompassing investigational Ru(III) or Ga(III) compounds. The majority of the reports deal with the analysis of biofluids (urine, plasma, pUF, etc.) that can be easily obtained from patients, whereas sampling of tissues is a more exhaustive. As a matter of fact, ICP-MS is particularly well suited for determination of the high mass elements, such as Pt or Ru, providing low LODs ( ng L-1) with little spectral interference. For the low mass elements (e.g. gallium), the analysis can be more problematic, because various matrix-related polyatomic interferences and high matrix level are possible (Filatova DG et al. Anal. Bioanal. Chem. 400 (2011) 709). Another inherent advantage of ICP-MS, the multi-element analysis capability, is rarely exploited.

With respect to interferences, it should be emphasized that ICP-MS analyses of biological samples are often difficult and require careful development of a sample preparation procedure. Two different approaches can be chosen for handling biofluid samples. The simplest one implies sample dilution with an appropriate diluent that has to ensure that in the final solution the drug remains in its original form. Alternatively, a digestion with concentrated nitric acid and hydrogen peroxide can be applied with the effect of reducing interfering organic molecules and bringing the metal ions into ionic form.

Being basically element-specific, ICP-MS lacks selectivity with regard to the different species of a given metal present in the sample. A separation procedure is commonly incorporated for the removal of unbound drug prior to the metal-determination step or the isolation of protein bound fraction from the excess drug in order to characterize a given metallodrug protein adduct.

Fractions are usually obtained from plasma by ultracentrifugation or ultrafiltration (Fig. 1). pUFs are most often characterized by the ICP-MS method. Typically, pUFs are obtained by centrifuging the plasma through a 10 or 30 kDa cut-off filter. The amount of metal in pUF, corresponding to free drug, usually varies in the range of 1020% for different metallodrugs and correlates with the total amount of drug present in blood (the rest of the drug is bound to proteins). Therefore, the pUF sample is considered to be representative for monitoring of metallodrugs in the course of pharmacokinetic, pharmacodynamic, and metabolism examinations.

Figure 1. General scheme of sample preparation implemented for measuring the distribution of a metallodrug in different blood fractions by ICP-MS technique. MAAD = microwave-assisted acid digestion (from Timerbaev AR et al. Trends Anal. Chem. 30 (2011) 1120).

Regardless of the aim of an analytical procedure, its validation should be of special concern.

As a matter of fact, plasma matrix components can interfere significantly with different metal isotopes related to a drug (or an internal standard). The impact of organic constituents and salts on nebulization efficiency and fluctuations of energy in plasma source can alternatively be reduced by microwave-assisted acid digestion of blood samples. Certified reference materials, while widely utilized for validation of ICP-MS methods intended on the determination of total metal concentrations, are fairly scarce in the case of biological material containing Pt, Ru or Au.

Therefore, there is a clear need for developing commercially available reverence material, ideally certified for both total and species concentrations, to support validated metallodrug analysis.

As a confirmation of the potential of ICP-MS for screening bloodstream transformations after administration, a comparative study of the affinity of Pt drugs to serum proteins, the results of which are shown in Fig. 2, could be mentioned. As expected, the highest affinity was measured for cisplatin (about 80% of Pt in the protein fraction) followed by carboplatin and oxaliplatin. High content of oxaliplatin (33%) was also found in the fraction containing erythrocytes, while 57% of free carboplatin was present in pUF in contrast to other drugs.

Figure 2. Metal content in various blood compartments after ex vivo incubation with platinum drugs Figures in bars show the average percentage of Pt (n = 3) in individual fractions (black for I, grey IIa, white IIb) designated and obtained as in Fig. 1.

Measurement uncertainty was 15% (adapted from Kapp T et al. J. Med. Chem. 49 (2006) 1182) A novel and not yet fully explored approach is due to the combination of ICP-MS with laser ablation (LA) for imaging of metals in solid biological samples. LA-ICP-MS requires no extensive sample preparation, as the metal under scrutiny is ablated directly from the surface of a sample (by irradiating with a laser beam) and thus converted to a plasma state naturally suitable for subsequent detection. Using LA-ICP-MS, it was possible to quantify platinum along a single hair strand from a cancer patient, as can be seen in Fig. 3.

Figure 3. Concentration of platinum measured by LA-ICP-MS in a single hair strand of a patient received four doses of cisplatin (100 mg each) (Pozebon D et al. Int. J. Mass Spectrom. 272 (2008) 57) The development of ICP-MS as a standalone method over the last few years is focused on validation of the quantification procedures, the performance of which could still be improved by exploiting the specific isotope dilution method. Addition of an isotopically enriched standard of the same metal, as contains in the metallodrug, to the sample would indisputably improve the accuracy and precision of the assay and make the drug-development output of ICP-MS measurements more reliable. Sample introduction systems that could ensure a better sensitivity of the method by offsetting low sample consumption (ICP-MS is sensitive to the mass of analyte, not to its concentration!) are also highly welcome.

Molecular MS ESI-MS. The soft ionization process characteristic of ESI generates multiply charged species of proteins, as well as other biomolecules, while keeping the molecular ions largely intact.

Therefore, ESI-MS is extremely effective to investigate the interactions taking place between selected metallodrugs and one or a few isolated proteins in the incubated sample. The coordination bonds that are formed between the metal and the protein are typically strong enough to resist cleavage during ESI.

The merits and limitations of the ESI-MS approach in studying metallodrugprotein interactions are nowadays carefully delineated and understood. The formation of drugprotein adducts may be straightforwardly assessed and precise information obtained, concerning the actual metal-to-protein stoichiometry. Even more importantly, careful analysis of the position of the ESI MS peaks allows identification of the chemical nature of the protein-bound metallic fragments.

Furthermore, time-dependent ESI-MS measurements are apt to monitor the kinetics of adduct formation, at least in those cases where the binding process is not too fast. While in early years, ESI-MS investigations with few exemptions encompassed metallodrug binding to serum proteins, more recently, attention has shifted to cellular proteins (most frequently, cytochrome c) that may constitute relevant biomolecular targets for metal-based drugs. It was shown that ESI-MS may be profitably employed to comparatively investigate the reactivity of structurally related metallodrugs (both classical and non-classical Pt compounds) toward the same protein or vice versa, a single drug interacting with different proteins. As demonstrated in Fig. 4, cisplatin, its inactive isomer transplatin, as well as carboplatin and oxaliplatin exhibit a roughly similar pattern of reactivity toward cytochrome c, dominated by the formation of monometallated adducts. In order to identify the protein binding site(s) for the metal, a specific procedure based on the tripsinisation of the proteindrug adduct can be implemented.

Figure 4. Deconvoluted ESI-MS spectra of (A) cytochrome c and its adducts with (B) cisplatin, (C) transplatin, (D) carboplatin, and (E) oxaliplatin (Casini A et al. ChemMedChem 1 (2006) 413) The advent of nano-ESI mode represents another recent methodological advancement that has further extended the usability of ESI-MS in the analytical mass-spectrometric laboratory. One of the remarkable features of nano-ESI is its extremely low sample consumption: only a few L of sample solution (with analyte concentration down to 108 M) are sufficient for molecular-mass determination and structural characterization. In addition, nano-ESI is something more than just a minimized-flow ESI since the low solvent flow-rate also affects the mechanism of ion formation.

ESI-MS can also be used for the characterization of drugprotein adducts directly in clinical samples. However, since the biomatrix (erythrocyte) constituents strongly suppress the ESI signal, resulting in low quality mass spectra, the analysis was only feasible after a rather tedious clean-up procedure, consisting of sample centrifugation, dilution and filtration of through a 10 kDa mass cut off filter to remove the low molecular weight fraction. Figure 5 depicts good quality mass spectra from which the hemoglobin adducts, containing from 1 to 4 platinum moieties, were identified in hemolysates taken one hour after administration.

Figure 5. ESI mass spectra of a hemolysate from a cancer patient under oxaliplatin treatment.

The peaks labeled with 14 represent hemoglobin bound to the respective number of Pt functionalities (Mandal R et al. Rapid Commun. Mass Spectrom. 20 (2006) 2533) However, it must be kept in mind that most of the present studies are performed on relatively small proteins because of the intrinsic difficulties of the method to deal with large proteins. Another major disadvantage is that this technique still cannot analyze real-life biological samples very well, as the intensity of the analytical signal is strongly affected by sample matrix.

Also, common buffers used in manipulations of such samples are to be avoided as they heavily perturb ESI-MS spectra. Finally, the multiple charges that are attached to the molecular ions may originate confusing spectral data.

MALDI-MS. Matrix-assisted laser desorption/ionization (MALDI) is another soft ionization technique pertinent to the MS analysis of biomolecular interactions for metallodrugs. MALDI-MS allows for assaying drug adducts, favorably formed by larger proteins, which tend to be fragile when ionized by ESI. This is due to use of a matrix (e.g. sinapinic acid) that protects the biomolecule from being destroyed by direct laser beam and facilitate vaporization and ionization.

However, since 2007 when the potential of MALDI-MS was for the first time evidenced (research groups of Dyson and Coling), only a few further studies exploited the MALDI approach.

To conclude, a variety of information that can be extracted from molecular MS experiments is distinctly wide. Therefore, further optimization and the development of precise experimental protocols adapted to each specific metallodrugprotein combination would increase the role of molecular MS techniques in understanding the behavior of these drugs in the organism.

SEPARATION-BASED (HYPHENATED) MASS-SPECTROMETRIC TECHNIQUES The presence of metallodrugs in biological systems in a variety of metabolic forms is the major reason why techniques with good separation power are required for analyzing their speciation. Therefore, any study concerning the biotransformation of drugs presumes hyphenation of direct elemental or molecular MS methods, with robust separation techniques like CE or HPLC.

Capillary electrophoresis There is a range of assets due to which CE is regarded as a superior technique compared with other separation-based methods. Although coupling of CE to ICP-MS is not simple, it is worth pursuing due to its benefits of an alternative to HPLC separation principle with higher separation efficiency, shorter analysis times, lower sample and buffer consumption but conceivably most important, milder separation conditions. The latter asset helps avoid undesirable compositional changes of analyte species in a CE system. On the other hand, the main challenge is the interfacing between CE and ICP-MS which is far from trivial, making CE-ICP-MS a task yet for specialists.

Today, at least two interfaces are commercially available, both adapting the low flow of CE (nL min1) to the higher flow (510 L min1), required to maintain stable performance of the interface system, by addition of a sheath flow. Another shortcoming of the technique is low sensitivity compared to HPLC-ICP-MS. This is primarily because of limited loading capacity of separation capillary that affords only nL injections, which limits the absolute amount of analyte entering the ICP-MS. Also, intraday changes in sensitivity causing precision to go beyond acceptable level are a nasty challenge. Two ways of circumventing this problem have been proposed, implying the addition of an internal standard to the sample (Mller C et al. J. Anal. At. Spectrom. 24 (2009) 1208) or an external standard to the sheath liquid (Groessl CG et al. Electrophoresis 29 (2008) 2224), both approaches leading to improved reproducibility.

ICP-MS interfaced on-line with CE serves as an especially useful tool for the characterization of metallodrugprotein interactions. A metal-specific nature of ICP-MS offers the possibility of distinguishing the signals of the intact drug and its protein-bound form(s), as well as some other possible metabolic forms, and hence specific monitoring changes in the metal speciation (Fig. 6). Recent applications of CE-ICP-MS include evaluation and comparison of the transport protein reactivity of ruthenium and gallium drugs (often in terms of rate constants), determination of the binding constants for the resulting adducts, and their resistance to speciation changes under simulated extra- and intracellular conditions.

Figure 6. Separation of platinum species induced by interaction of cisplatin with human serum albumin. Peaks: (1) cis-diammineaquachloroplatinum(II);

(2) cisplatin;

(3) cisplatin-albumin adduct (Timerbaev AR et al. Electrophoresis 25 (2004) 1988) Nonetheless, to date only a few CE-ICP-MS applications have been progressed to real biofluid circumstances, most of them directed on elucidation and quantitative characterization of protein-mediated profiles of metallodrugs in human serum. In the most advanced application, the method was brought up to the analysis of serum from a patient treated with a developmental Ru(III) drug, which showed that the drug is mainly bound to albumin and only marginally to other serum proteins (Fig. 7), taking advantage of multi-elemental detection capability of ICP-MS (notice the traces of 34S and 102Ru isotopes in the figure). Another recent study (Abramski JK et al. Analyst (2009) 1999) aimed on shedding new light onto the fate of oral gallium drug upon entering the bloodstream, revealed that the metal profiling recorded by CE-ICP-MS in human serum generally resembled that of individual transferrin binding.

Figure 7. CE-ICP-MS analysis of human plasma samples taken on days (B) 2 and (C) from the beginning of repeated intravenous administration of the Ru-based drug.

Trace A shows in vitro drugs binding to transferrin and albumin (peaks 1 and 2, respectively) (Groessl CG et al. Electrophoresis 29 (2008) 2224) Summarizing, much progress has been made in implementing the ICP-MS-based metallodrugproteomics assays, supported by CE separation. The significant trend appears to be the advent of CE-ICP-MS in exploring the binding of metallodrugs to proteins under biological-fluid and cytosol circumstances.

However, until application of sensitive mass detectors, such as high resolution sector-field ICP-MS, comes true, real-sample analyses will be troublesome suffering from a lack of sensitivity, most critical for less abundant binding partners. The response might arrive both from the side of manufacturers (e.g., through proposing interfaces with better fluid dynamics and a minimized dead volume than those of currently available commercial set-ups) and due to efforts of CE method developers toward offering an assay combined with an in-line preconcentration step. Also, the range of applicability of CE in the area could benefit from isotope dilution and collision/reaction cell methodologies that have already become workable in metalloproteomic analysis by CE-ICP-MS.

Although the coupling of CE to ESI-MS has been launched earlier and advanced further than that of ICP-MS, this hybrid method has not been employed in biofluid analysis of metallodrugs at the time of writing.

High-Performance Liquid Chromatography The coupling of an ICP-MS detector to a HPLC system is much more straightforward, as the characteristic flow rates of the two techniques are readily compatible (0.11.0 mL min 1).

However, there is a requirement of special consideration to meet in the development of an apt HPLC-ICP-MS procedure. As metal-based drugs are with few exemptions lipophilic substances, the common separation principle is reversed-phase HPLC rested on using organic solvent-containing eluents. However, the ICP is not very tolerant toward organic solvents that cool the plasma and change ionization characteristics, leading to decreased sensitivity. Applying gradient elution, for which the content of an organic modifier is varied during the chromatographic run, poses a special challenge because of concomitant sensitivity alterations that compromise quantification. Several approaches for minimizing the influence of organic solvents on the ICP-MS sensitivity have been attempted. The plasma load can be reduced either by decreasing the eluent flow-rate, or cooling the sample aerosol and thereby removing the organic solvent, or by its complete removal before introduction to the detector. Note that this matter is much less troubling when size-exclusion chromatography (SEC) is in use, which eluents are essentially aqueous buffer solutions (e.g. Tris buffers) and do not alter significantly the sensitivity of ICP-MS.

SEC-ICP-MS has a proven ability of separating the low molecular mass drugs from the high molecular mass protein adducts. One disadvantage of SEC is that the peaks are often broad (see Fig.

8). Nonetheless, separation of the free drug and the major protein-bound fractions typically presents no difficulty, even though the efficiency is not optimal. As can be seen in Fig. 8, the protein fraction at 60 kDa (likely due to the drugalbumin adduct) is well resolved from the free drug fraction (2 kDa), whose exact identification is albeit not possible using SEC alone.

Figure 8. SEC-ICP-MS chromatogram of plasma after 1 h from the end of oxaliplatin injection.

Y-axis shows counts for m/z 195 (Allain P et al. Drug Metab. Dispos. 28 (2000) 1379) For biological fluids, sample pretreatment is well described and relatively simple. Plasma, pUF, and urine can be injected into an HPLC system directly (preferably, after filtration to protect the column) or after dilution with water or an eluent. Harsher procedures are needed for solid samples in order to transfer metallodrugs and their metabolites into solution.

A recent upgrading of HPLC-ICP-MS was due to utilizing detection systems equipped with collision/reaction cells. Many of polyatomic interferences, e.g., those due to 56ArO or 57ArOH for iron presented in transferrin, can be surpassed by using the collision or reaction gas. Moreover, the use of oxygen as a reaction gas results in interference-free detection of sulfur (owing to creation of the molecular ion, 48SO+) and thus affords a unique possibility to measure the metal-to-sulfur ratio in protein adducts. If the number of the amino acids containing sulfur is known, the adduct stoichiometry may be quite accurately evaluated.

Another up-do-date advancement concerns using 2-D chromatography, in which a size exclusion column and two small monolithic ion-exchange columns were interfaced with ICP-MS in an on-line arrangement. However, this approach was only advanced to mapping cisplatin interactions in serum (Fig. 9), since the recovery of ruthenium drugs from the size-exclusion column was below 80%.

Figure 9. 2-D chromatograms of fetal calf serum ex vivo incubated with cisplatin. Anion-exchange chromatograms shown in the upper insets were obtained on-line for the corresponding SEC fraction.

Peaks were identified as cisplatin adducts with PB1 albumin dimer;

PB2 transferrin, and PB3 albumin. (Heffeter P et al. J. Biol. Inorg. Chem. 15 (2010) 737) In recent years, the interest in application of micro-HPLC systems has also increased, as smaller columns with diameters less than 1 mm have become commercially available. When such columns are hyphenated with ICP-MS, the load of an organic solvent into the ICP is significantly reduced and sensitivity restored. The use of microcolumns in combination with ICP-MS will probably increase in the future, as in addition, these allow for the analysis of very small samples (e.g. 300 nL), an upright advantage when determining metallodrugs in small clinical samples such as biopsies or cell fractions.

HPLC-ICP-MS is a powerful tool for assaying the protein-binding behavior of metallodrugs, as it can in most cases provide adequate sensitivity for characterization of individual metal species in real-world samples. As such the method has found widespread application in the field since its introduction over 10 years ago, particularly in situations where efforts are made to eliminate interactions with the stationary phase and to attain the complete recovery of the compounds of interest. Most likely, this avenue will continue over the coming years, being supported by (i) improvements in accuracy and precision, permitting more reliable measurements (including stoichiometric ratios);

(ii) comparative studies on drug interactions with metal-binding proteins (through monitoring metal exchange);

(iii) implementation of on-line LC LC settings with a fast column switching for high-throughput screening assays, and (iv) the metal-peptide mapping that could benefit from added advantages of ICP-MS and ESI-MS couplings.

On-line coupling of chromatographic separation and molecular MS detection as realized in HPLC-ESI-MS is insofar limited to quite a few examples. The obvious explanation of less wide spread usage is the methods much poorer sensitivity compared to HPLC-ICP-MS, making it difficult to obtain good quality mass spectra at low analyte concentrations, especially for those analytes that are hardly ionized by electrospraying.

Nonetheless, in essence, HPLC-ICP-MS and HPLC-ESI-MS complement each other and when applied in combination, would provide a very powerful analytical methodology capable of detecting, identifying, and quantifying metallodrugs and their biotransformation products in biological samples. In this regard, the interested reader is referred to the first published account on simultaneous ICP-MS and ESI-MS detection, after splitting the effluent from the HPLC column, focused on a developmental Pt(II) drug in pUF (Smith CJ et al. Anal. Chem. 75 (2003) 1463).

Common for both hybrid techniques is that the limiting analytical factor is more often the efficiency of the separation side of hyphenation, occasionally suffering from broad, unresolved peaks or long runtimes. Thus, optimization of the separation appears as an important issue in further development of hyphenated MS methodology in the field.

CONCLUSIONS Mass spectrometry has an enormous impact on all fields of biomedical research, development and application of metal-based drugs being no exception. Indeed, straightforward analytical approaches are indispensible for the generation of knowledge on biotransformation routes of new drugs, with identification and possibly quantification of a multitude of metabolic forms, and on clinical pharmacokinetics and cell accumulation of established therapeutics, providing them a firm basis for the safe and effective use in the clinic. Despite significant instrumental and methodological improvements achieved in the past decade, there is still room to improve conventional MS methods and introduce alternative approaches, as was emphasized in the present lecture.

Acknowledgements Financial support of the Russian Foundation for Basic Research is gratefully acknowledged.

The authors also express sincere gratitude to the members to their joint team who participated in conducting original research and review work highlighted in this lecture, and personally to Profs.

Mikhail Bolshov, Maciej Jarosz, Luigi Messori, Katarzyna Pawlak, Stefan Strup, Drs. Jan Abramski, Darya Filatova, Chiara Gabbiani, Irina Seregina, and others.

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