1. XSeries II ICP-MS Clinical Applications

2. 2 Why is clinical analysis important? Some elements are toxic; occupational needs to be monitored -Al - causes dementia; critical element for renal patient monitoring -As - acutely toxic as inorganic As (vomiting, coma, death) -Cr - Cr(VI) carcinogenic when inhaled -Pb - leads to cognitive function impairment Some elements are essential, so deficiency causes ill-effects -Se - increased risk of cancer, heart disease -Fe - anaemia Some elements are essential at low levels, but toxic if present in excess -Se, Cu

3. 3 Requirements for clinical sample analysis Wide range of elements to be measured Large dynamic range –When major and trace results are required Detection limits in the ppt to ppb range in the original sample Rapid washout between samples High matrix tolerance –Blood, urine, serum and tissue digests Stable and robust sample introduction –Suitable nebulizer and torch design Na: blank to 300 ppm Cr: blank to 100 ppb

4. 4 Sample types to be analysed - what and why? (1) Concentration changes day to day and throughout the day; these changes reflect an individual’s diet and state of health Urine composition can aid medical practitioners in diagnosis of certain conditions Urine measurement can be used for monitoring of occupational exposures of workers in high-risk industries Salt content of urine is around 0.1% (m/v). Could be aspirated directly, but salt level can be higher (e.g if patient is dehydrated), so dilute it 1:10 with nitric acid to ensure matrix concentration is below the ICP-MS dissolved solids limit of 0.2% (m/v) ICP-MS detection limits for elements of interest are way below the levels found in urine, so dilution induces no analytical compromise Urine (95% water, 2.5% protein, 2.5% other materials)

5. 5 Sample types to be analysed - what and why? (2) Serum (0.7% protein, inorganic salts, water) Serum is the fluid that is separated from clotted blood or blood plasma that is allowed to stand Similar composition to plasma but lacks fibrinogen and other substances that are used in the coagulation process Serum is a useful medium in which to measure the body’s Cu, Zn and Se levels or for diagnosis and monitoring of certain diseases (e.g. Wilson's disease) and for nutritional studies Cannot be aspirated directly and usually only available in µl quantities, so at least 1:10 dilution applied prior to analysis Can use nitric acid for dilution, but customised clinical diluent preferred (maintains better long term sample stability)

6. 6 Sample types to be analysed - what and why? (3) Blood (90% water, 10% red / white blood cells / platelets plus proteins and inorganic salts) Useful medium for measuring occupational exposure (especially of Pb, Hg and Cd) - these analytes become associated with haemoglobin so are much higher in whole blood than in serum Cannot be measured directly; at least 1:10 dilution with either a customised clinical diluent or dilute ammonia solution required Cannot dilute with strong acid; blood cells and proteins precipitate Detection limits achievable with ICP-MS for Pb, Cd and Hg in blood are below the levels found, so dilution induces no analytical compromise

7. 7 Instrumentation used for clinical sample analysis Flame AAS –Detection limits not low enough for trace analysis –Ideal for rapid Na, K assays Graphite furnace AAS –More difficult to use than flame AAS –Time consuming and prone to contamination –Technique uses low sample volumes - ideal for some clinical analyses –Low detection limits ICP-MS –Fast, multi-element capability –Can measure sub-ml sample volumes –Low detection limits –Qualitative scan capability

8. 8 Potential problems when analysing clinical samples with ICP-MS Nebuliser and/or torch injector blockage –Use appropriate nebulizer (concentric, Burgener Miramist) –Use wide bore injector (2.0 mm) torch –Digest samples (can get contamination problems; large dilution factors degrade detection limits) Low sample volume (can be <1 ml) –Dilute sample (the simplest and preferred option) –Use low-flow nebulizer (may block with serum / blood) Matrix related interferences –Select another isotope free from interference –Matrix match standards –Use collision / reaction cell to eliminate the interference

9. 9 CCT ED – Interference Elimination Several clinically important elements suffer from polyatomic interference with quadrupole ICP-MS Can be removed using CCT ED with H 2 /He reaction gas with the X SeriesII Automated within-sample switching is provided between CCT and non-CCT elements - Optimum performance for ALL elements

10. 10 Sample preparation options / considerations Microwave or hotplate digestion –Contamination from digestion vessels, reagents and laboratory environment –Large dilution factors (to reduce acid concentration); degrades detection limits Sample dilution –Contamination in the diluent reagents –Loss (precipitation) of certain analytes in some diluent matrices e.g. ammonia –Cannot dilute blood with acid (cellular material aggregates and precipitates – analyte loss and possible nebulizer blockage) Solutions to these issues….. –Pre-clean digestion vessels; check digestion blanks before preparing samples –Use high purity reagents and wear powder free gloves while handling samples –Use a diluent reagent containing a complexing agent (e.g. EDTA) and/or trace nitric acid (0.05%) to maintain analytes in solution - PREFERRED OPTION

11. 11 Sample preparation - proposed clinical diluent Need clean diluent that keeps analytes in solution and matrix matches standards with samples; must keep acid concentration low for blood samples Diluent componentPurpose 3% (w/v) butanol Matrix matches sample and standard carbon content to overcome ionisation enhancement effects on As and Se 0.05% (v/v) HNO 3 Stabilizes metallic analytes in solutions 0.01% (v/v) TMSC Stabilizes diluted blood and serum samples as an emulsion) 200ppb Au Stabilizes Hg in solution (as Hg 2+ ), due to the Au 3+ / Hg 0 reaction (E=+0.65V) Ga, Ge, Rh, Te, Re, Tl, Bi Appropriate low, mid, high mass internal standards for clinical samples Proposed general clinical sample diluent Ge and Te preferred internal standards for higher ionisation potential elements such as Zn (Ist ionisation potential of Ge = 7.9eV, Zn = 9.4eV and Te = 9.0eV

12. 12 CCT & Normal mode calibrations and precision using clinical diluent 80Se111Cd52Cr Isotope9Be27Al52Cr55Mn59Co63Cu75As80Se111Cd208Pb Acquisition modeStdCCT StdCCT Std Concentration (ppb)1411141411 Mean cps735530144985966391815167223948838989470 RSD (%) (n=3)1.71.91.80.30.70.80.7 1.81.0 Stability of in-sample switching - standard to CCT mode (8% H 2 /He collision gas)

13. 13 Analysis of blood and serum using clinical diluent UK NEQAS samples analysed –Mixed batch analysis: - 12 whole bloods (for Cd and Pb) and 10 serums (for Cu and Zn) Seronorm reference materials also measured –Seronorm serum –Seronorm blood level 1 and 2 Two-point calibration solutions prepared in the diluent matrix Burgener Mira Mist nebulizer, quartz Peltier cooled impact bead spray chamber, standard injector diameter torch and PlasmaScreen Plus (for enhanced sensitivity) used Standard instrument operation used (i.e. without collision cell employed) Total analysis time (uptake, wash and 3 repeats / sample) = 3 min –Qualitative survey run across full mass range also included

14. 14 Serum analysis results, Cu Seronorm serum Average Cu relative accuracy to consensus results = 92%

15. 15 Serum analysis results, Zn Seronorm serum Average Zn relative accuracy to consensus results = 94%

16. 16 Blood analysis results, Pb Seronorm blood level 2 Seronorm blood level 1 Average Pb relative accuracy to consensus results = 101%

17. 17 Blood analysis results, Cd Seronorm blood level 2 Seronorm blood level 1 Average Cd relative accuracy to consensus results = 105%

18. 18 Example applications Fast analysis of Pb and Cd in whole blood reference materials and UK NEQAS samples Measurement of Hg in urine Measurement of Al in clinical samples Sample analyses performed using a Thermo Electron XSeries II ICP-MS

19. 19 Fast Cd and Pb in whole blood analysis Seronorm and Bio Rad blood certified reference materials measured together with two NEQAS reference samples All samples diluted 1:50 with 0.1% ammonia, 0.01% Triton X-100 solution Rh (10ppb) used as internal standard Standard additions calibration used; 1% HNO 3 blank, low Pb / Cd level blood zero addition standard, then: –1, 2.5, 5,10, 20ppb Cd and 100, 200, 400, 600, 800 ppb Pb addition standards Burgener AriMist nebulizer, with standard injector diameter torch and PlasmaScreen Plus (for enhanced sensitivity) used Autosampler probe-to-wash-early function used to maximise throughput Total analysis time (uptake/wash and 3 repeats / sample) = 1.2 min Equivalent to 51 samples per hour

20. 20 Cd and Pb calibrations and figures of merit 111 Cd Parameter 111 Cd 208 Pb Sensitivity (cps/ppb)321030800 1% HNO 3 blank BEC (ppb)0.0080.237 Detection limit (3σ, n = 5) from this calibration (ppb)0.0040.005 Correlation coefficient0.999930.99991 208 Pb

21. 21 Fast Pb & Cd in blood results Good agreement achieved between the measured and reference values Sample identityMeasured 111 Cd result (ppb)* Expected 111 Cd result (ppb) Measured 208 Pb result (ppb)* Expected 208 Pb value (ppb) Seronorm blood 2 6.2 ± 0.55.4 - 7.2412 ± 13353 - 443 Seronorm blood 3 10.3 ± 0.310.6 - 11.5702 ± 6611 - 687 Bio Rad 1 4.5 ± 0.13.4 - 5.194.1 ± 0.874 - 112 Bio Rad 2 12.5 ± 0.410.4 - 15.6254 ± 2230 - 340 Bio Rad 3 33.3 ± 0.526 - 39499 ± 1430 - 650 NEQAS 608a 9.86 ± 0.0810.5203 ± 1217 NEQAS 608b 3.9 ± 0.24.1471 ± 2483 NEQAS 609a 13.1 ± 0.313.27.0 ± 0.2not reported NEQAS 609b 0.82 ± 0.041.1374 ± 4397 1:50 dilution corrected data shown All data in ppb, ± 1 std. dev.

22. 22 Measurement of Hg in urine Why measure Hg –Toxic metal (especially in Hg 2+ and organo- mercury forms) –Hg 2+ causes: Erythrism (behavioural abnormalities (Mad Hatter syndrome)) Stomatitis (inflammation of the mouth lining) Kidney damage and uraemia (build up in the blood of products normally excreted in the urine) –Workers in certain industries suffer significant occupational exposure to Hg Caustic soda and chlorine production Gold refining

23. 23 Analysis details 5 samples supplied - diluted (1:10) with 2% (v/v) HNO 3 containing 200ppb Au (to stabilize Hg) and Bi internal standard (2ppb) added Calibration solutions prepared at blank, 1, 5 and 10ppb levels Isotopes measured: 200 Hg, 202 Hg, 209 Bi Spike recovery tests performed and relative accuracy of the method assessed using Seronorm urine Glass concentric nebulizer, quartz Peltier cooled impact bead spray chamber, standard injector diameter torch and PlasmaScreen Plus (for enhanced sensitivity) used Cannot use GF-AAS directly for analysing Hg (volatile analyte so lost in ashing step) - need to use vapour generation method

24. 24 Hg calibrations and figures of merit 200 Hg 202 Hg Parameter 200 Hg 202 Hg Sensitivity (cps/ppb)54947132 BEC (ppb)0.1930.192 Limit of detection (3σ, n = 10) (ppb)0.011 Correlation coefficient0.999970.99993

25. 25 Analyte Short (10 minutes, n = 20) RSD (%) Medium (4 hours, n = 23) RSD (%) 200 Hg0.81.9 202 Hg0.71.9 209 Bi0.41.6 Short / medium term stability for Hg in urine Without internal standard correction With internal standard correction Analyte Short (10 minutes, n = 20) RSD (%) Medium (4 hours, n = 23) RSD (%) 200 Hg0.61.8 202 Hg0.61.9 Internal standard signal suppression due to urine matrix = 27%

26. 26 Hg washout from the system Hg washes down to background after 60 seconds Time resolved analysis trace from blank to 10ppb Hg and back to blank Both solutions spiked with Au at 200ppb

27. 27 Sample results and spike recoveries Sample identity Result (ppb)Spike result (ppb)Recovery (%) Sample 1 6.95 ± 0.161.79 ± 0.0189 Sample 2 6.81 ± 0.101.86 ± 0.0292 Sample 3 5.21 ± 0.171.94 ± 0.0196 Sample 4 4.19 ± 0.092.08 ± 0.04103 Sample 55.65 ± 0.251.71 ± 0.0586 Reference value (ppb) Seronorm urine48.9 ± 0.248 ± 2102 Acceptable recoveries obtained from the Hg spiked samples Good agreement obtained between measured and reference Hg values in Seronorm urine

28. 28 Measurement of Al in clinical samples Why is Al important? –Chronic exposure to elevated levels of Al known to cause dementia (memory loss, impaired cognitive function) (distinct from Alzheimer's disease) –Individuals with kidney failure accumulate Al in the blood – renal patients at risk What samples types must be measured? –Reverse osmosis water –Dialysis fluids and serum

29. 29 Measurement of Al - Analysis details 6 water samples, 5 dialysis fluid solutions, 2 certified human sera materials (UTAK) and 4 bovine sera (measured in duplicate) supplied Water and dialysis fluids diluted 1:10 and serum samples diluted 1:20 with 2% (v/v) HNO 3, Be and Ga internal standards added Calibration solutions prepared at blank, 1, 5, 10 and 20 ppb Isotopes measured: 9 Be, 27 Al, 71 Ga Spike recovery tests (5ppb Al added) performed Relative accuracy of the method assessed using the UTAK reference sera Burgener Miramist nebulizer, quartz Peltier cooled impact bead spray chamber, standard injector diameter torch and PlasmaScreen Plus (for enhanced sensitivity) used Total analysis time (uptake, wash and 3 repeats / sample) = 2.5 min –Qualitative survey run across full mass range also included

30. 30 Aluminium calibration and figures of merit 27 Al Parameter 27 Al Sensitivity (cps/ppb)7498 BEC (ppb)0.334 Detection limit (3σ, n = 5) from this calibration (ppb)0.015 Correlation coefficient0.99997

31. 31 Water and dialysis fluid results (all data in ppb) Sample identity Measured value (± 1 sd)Consensus value Added amount Water 1925 62.68 ± 0.6363.87 ± 6.96 60 Water 1926 153.8 ± 0.77150.19 ± 24.36 150 Water 1929 12.06 ± 0.2313.7 ± 3.52 10 Water 1930 40.79 ± 0.3742.08 ± 12.66 40 Water 1933 170.6 ± 0.1177.11 ± 29.96 170 Water 1934 233.6 ± 0.6228.98 ± 39.10 220 DF 1912 12.32 ± 0.1216.56 ± 9.66 10 DF 1919 103.7 ± 0.52104.33 ± 30.11 100 DF 1920 48.24 ± 0.0451.05 ± 16.99 40 DF 1935 96.67 ± 0.34107.02 ± 27.05 90 DF 1936 68.55 ± 0.2878.84 ± 39.29 60 Good agreement achieved between the consensus and X SeriesII results and data consistent with the known added amount of Al

32. 32 Serum sample results (all data in ppb) Acceptable agreement achieved between the consensus/reference data and the X SeriesII results (except sample H (ICP-MS data lower)) Sample identityMeasured valueConsensus / reference value Serum E1 15.81 ± 0.2714.30 ± 3.24 Serum E2 14.94 ± 0.4314.30 ± 3.24 Serum F1 36.06 ± 0.1439.66 ± 4.58 Serum F2 40.21 ± 0.9039.66 ± 4.58 Serum G1 65.11 ± 1.0364.49 ± 6.21 Serum G2 67.55 ± 1.2664.49 ± 6.21 Serum H1 77.66 ± 1.4493.09 ± 7.83 Serum H2 84.16 ± 0.5593.09 ± 7.83 UTAK lot #: 4103 14.65 ± 0.4717 ± 7 UTAK lot #: 4808 192.5 ± 2.3215 ± 43

33. 33 Spike recovery test results (all data in ppb) Quantitative spike recoveries obtained for all three sample types Sample identityMeasured result*Spike resultSpike recovery (%) Water 19256.27 ± 0.0611.33 ± 0.03101 Water 193423.36 ± 0.0628.31 ± 0.0299 DF 19121.23 ± 0.016.56 ± 0.04107 DF 19366.86 ± 0.0311.84 ± 0.03100 Serum E10.79 ± 0.015.78± 0.04100 Serum H24.21 ± 0.039.57 ± 0.09107 * before dilution correction

34. 34 Conclusions The XSeries II ICP-MS is ideally suited to clinical analysis –Low detection limits, multi-element capability, wide dynamic range –Faster, simpler and more flexible than GF-AAS –Easily achieves the required performance for routine assays in blood, serum and urine –Offers survey scan option for every sample - enables qualitative and semi-quantitative evaluation of analytes that have not been measured fully quantitatively Blood / serum samples analysed in a single run after simple dilution step Optimised configuration developed for general clinical sample analysis –XSeriesII ICP-MS + CCT, Burgener Miramist nebulizer, PlasmaScreen Plus –Wide bore injector torches available (if required) to prevent injector blockage Collision cell technology offers improvements in clinical sample analysis –Ar-based interferences no longer a major limitation for accurate determination of Cr, As and Se at trace levels