Determination of Essential, Therapeutic. in Biological Materials; Applications of

Transcription

1 ESAC Copenhagen 15 th April 2008 Determination of Essential, Therapeutic and Toxic Elements and Their Compounds in Biological Materials; Applications of ICP-MS in the Clinical i l Laboratory Ed McCurdy, ICP-MS Specialist, Agilent Technologies Ltd

2 Scope Introduction to ICP-MS for clinical sample analysis Requirement for elemental analysis in clinical samples Routine trace element monitoring Analysis of toxic and harmful elements Elemental screening, poisoning, radionuclides Toxic organometallic compounds ICP-MS analysis for the measurement of organic compounds Identification and quantification of pesticide residues Quantification of Chemical Warfare Agent degradation products Forensic Applications of ICP-MS Laser Ablation ICP-MS Direct Analysis of Elemental Distribution in Tissues Page 2

3 What is ICP-MS? An inorganic (elemental) analysis technique ICP - Inductively Coupled Plasma MS - Mass Spectrometer high temperature electrical discharge, which decomposes, atomizes and ionizes samples forms ions, so compounds not measured directly quadrupole ( quad ) mass analyzer mass range from 5 to 260 amu (Li to U...) separates all elements in rapid sequential scan isotopic information available ions measured using dual mode detector ppt level LODs for most elements Calibration range up to 1000 s ppm Spectral interferences removed using collision/reaction cell Page 3

4 Agilent 7500cx ICP-MS System with Collision/Reaction Cell (CRC) Reaction Gas Inlet High temperature 27MHz plasma generator Multi-element interference removal by on-axis octopole reaction cell Fast simultaneous dual mode detector (9 orders dynamic range) Plasma Low flow sample introduction system Off-axis Lens Octopole High frequency hyperbolic quadrupole Page 4

5 Elements of Interest in Easily Extractable Fluids (Urine, Blood, Serum, Plasma) Highly toxic heavy metals As, Cd, Pb and Hg Potentially toxic elements Al, Sb, Ba, Be, Bi, Li, Ni, Sr and Tl Essential elements Cr, Co, Cu, Mg. Mn, Se, V and Zn Wide range of elements may be measured, including many, such as Be, As, Se, Hg, U, which are considered difficult by other techniques Page 5

6 Which Elements can be Measured Using ICP-MS? All elements in colour can be measured only those elements present in the plasma gas, <5amu, >260amu, and those which are not ionized, are inaccessible Page 6

7 Analytical Needs for Inorganic Measurements in Clinical Laboratories The technique must possess robust sample introduction To handle large sample numbers and differing matrices routinely monitored Measurement of many elements in the same fast acquisition Removal of interferences Screening applications Sample turnaround/productivity Reduced cost of analysis Quadrupole ICP-MS can meet all of these criteria Make measurements at trace levels (low detection limits) and at high concentrations in the same acquisition To reduce reruns, improve productivity and lower costs The ability to measure element species is also provided by ICP-MS Expands range of applications, and provides useful background research info Page 7

8 Collision/Reaction Cell to Remove Matrix-Based Spectral Interferences Apparent Cr5 52 Concentrat tion in Blank (ppb) Cr52 ClO/ClOH (mass 52) overlap in HCl matrix AC( ArC (mass 52) overlap in acetic acid matrix ArC and ClO overlaps in combined matrix 01% 0.1% 5% 5% HCl 1% 1% 200ppm 200ppm 500ppm Mixed HNO3 HNO3 H2SO4 AcOH Na Ca P Matrix H2 He NoGas Advantage of Collision/ i Reaction Cell ICP-MS Provides removal of spectral overlaps allows accurate trace element analysis in variable high-matrix samples Example shows the removal of ClO, ArC interferences in various matrix blanks He collision mode gives reliable removal of all polyatomic interferences regardless of the sample matrix composition Matrix Blank Page 8

9 Routine Analysis of Urine Sample Preparation and Calibration Direct analysis of urine samples following a 1/5 or 1/10 (v/v) dilution with deionized water and nitric acid No clogging of the nebulizer No particle deposition in the injector tube over 12 hours of analyses Calibration using Method of Standard Additions (MSA), so matrix-matched standards Standard addition calib is then converted to external calib and applied to subsequent samples 5 µg/l Tb internal standard added to all sample and calibration solutions Agilent Application Note: Rapid and reliable routine analysis of urine by Octopole Reaction Cell ICP- MS, EN, by Peter Heitland, Medical Laboratory Bremen, Germany Page 9

10 Measured and Certified Concentrations in Urine Reference Material Lyphochek Element Concentration (µg/l - ppb) Lyphochek, level 1 Lyphochek, level 2 Measured certified Measured (n=10, external) (n=10, external) certified Cr 1.7 ± ± ± ± 4.1 Co 6.6 ± ± ± ± 4.2 Cu 24 ± ± ± ± 10 Se 56 ± ± ± ± 37 As 65 ± 6 67 ± ± ± 33 Cd 8.4 ± ± ± ± 3.1 Sb 6.9 ± ± ± ± 7 Tl 96± 9.6 ± ± 9.7 ± ± ± 40 Pb 13.5± ± ± 5 69 ± 14 Data: Medical Laboratory Bremen, Germany Page 10

11 High Throughput Analysis of Blood Samples using 7500ce ICP-MS Biomonitoring of trace elements in human blood samples - an important tool for occupational and environmental health Goals of this study Determine a high number of trace metals in blood of 130 unexposed subjects Develop a rapid routine method for the multi-element analyses of blood using collision/reaction cell-icp MS Blood samples were collected in lithium heparin monovettes 500 ul of the sample was diluted with 100 ul 0.1% (v/v) Triton-X-100 solution and 500 ul of the internal standard solution This solution was made up to 5 ml with a 0.5% (v/v) NH 4 OH solution in a 10 ml polypropylene autosampler tube More than 100 samples can be prepared in less than 1hour by one person Biomonitoring of 37 trace elements in blood samples from inhabitants of northern Germany by ICP MS, Peter Heitland, Helmut D. Koster, Journal of Trace Elements in Medicine and Biology 20 (2006) Page 11

12 Analytical Figures of Merit for Blood Analysis Limits of quantification (LOQs), calculated in undiluted blood, range from ug/l for 238 Uto01ug/L 0.1 for 69 Ga Spike recoveries of 1 ug/l (10 ug/l for B, Mn and Sr; 200 ug/l for Cu and Rb) from single element calibration solutions are in the range % Data: Peter Heitland, Helmut D. Koster, Medical Laboratory Bremen, Germany Page 12

13 Effects of Sample Matrix on the Sample Introduction System Sample Cone Skimmer Cone Photos of the interface and sample introduction ti system after a 90-sample run (whole blood). Both the sampler and skimmer cones show only minor matrix deposits none at the cone tips The standard 2.5mm injector torch used was virtually depositfree. The blood deposits on spray chamber and the nebulizer block were removed using a sodium hypochlorite solution Images: R. Wahlen et al., LGC Limited, UK Page 13

14 Screening for Toxic and Harmful Elements Elemental Screening of 1:10 diluted Urine (with interference removal in He mode) Screening to identify poisons (1:10 diluted urine scan) Unique Unknown capability element of spiked ICP-MS into acquire urine sample a scan across the entire mass range in about 2 minutes, screening elements from 1000 s ppm to sub-ppb levels [1 ] S p e c tru m N o.1 [ s e c ]: S M P L.D # / T u n e # 1 [C P S ] [L in e a r] 1.0 E 5 C Na Rb Mg 5.0 E 4 Unknown element Li CaC a Cu Fe Zn Br As Mo Sr I Sb Cs Sn Ba Pb m / z-> > Page 14

15 Analysis of Toxic and Harmful Elements Screening to identify poisons (1:10 diluted urine scan) Confirmation (from isotopic template) of presence of Thallium (2ppb spike) Can be quantified (semiquant) by reference to known concentration element Note 210 Po would also be seen in this mass region of the screening acquisition [1 ] S p e c tru m N o.1 [ s e c ]:0 005SMPL.D# 3 S M P L / T/ u n e # 1 [C P S ] [L in e a r] 1.0E5 Alexander Litvinenko - poisoning 5.0E4 205 Tl Tl 203 Tl 210 Po 208 Pb Pb m/z- > Page 15

16 Measurement of Radionuclides Left: Calibration for radionuclides is easily achieved at sub ng/l (ppt) levels, even with standard sample introduction (U used for illustration) Radionuclides are typically easily ionized and the spectrum is free from overlaps and backgrounds, so LOD s in the pg/l (ppq) range are achieved Scan of 1ppt 237 Np standard, showing high sensitivity and low random background Page 16

17 Radionuclide Analysis Quantification and Isotope Ratio Measurement Very high sensitivity and low background for 10ppt U in 1:10 diluted urine ~1.2 million cps/ppb U Natural U spike, so 235 U (0.72% abundance) concentration was 72ppq 2.0E4 [1] Spectrum No.1 [ sec]:10urine.d / Tune #1 [CPS] [Linear] 238 U (99.27%) U ICP-MS also provides isotopic information, so U isotopic pattern (isotope ratio) can be used to identify source of contamination Natural = 0.72% 235 U Waste depleted = % 235 U Pile depleted ~0.6% 235 U Enriched >0.72% 235 U 1.0E4 235 U (0.72%) m/z-> Page 17

18 Toxic Elemental Forms or Species For many elements, the level of toxicity is highly dependent on the chemical form of the element, so separation (chromatography) is required GC Laser Ablation ICP-MS LC Optional Conventional Detector(s) e.g. ESI-MS CE % P Respo onse (CPS) m/z Time (min) Page 18

19 Example: LC-ICP-MS for As Speciation Toxic! Less-Toxic Non-Toxic? Many As species exist the inorganic As species are known to be toxic and most organic species are relatively l harmless to humans. The potential toxicity of some species, such as the huge variety of arsenosugars, has not yet been established. Courtesy Ute Kohlmeyer GALAB, Germany Page 19

20 Chromatogram of As Standard (1.0 µg/l each) A new column has been developed to provide routine separation of the 5 most common As species in urine: Column G (4.6 x 250 mm) g pp Guard Column G Mobile Phase (Basic): 2 mm phosphate buffer solution (PBS) ph 11.0 adjusted with NaOH 02mMEDTA mm, CH 3 COONa 3.0 mm NaNO 3 1% ethanol Agilent Application Note: Routine Analysis of Toxic Arsenic Species in Urine Using HPLC with ICP-MS, EN, by Tetsushi Sakai and Steven Wilbur, Agilent Technologies Page 20

21 Determination of Organo-As Species Using HPLC with ESI-MS & ICP-MS ICP-MS Agilent 7500 HPLC Agilent % 85 % ESI-MS Agilent 1100 Elemental specific detection Molecular specific detection Single HPLC System with controlled split to provide sample flow to ESI-MS and ICP-MS. Provides simultaneous measurement of As-containing compounds and As concentration (ICP-MS) and concentration/structural information on the organic part of the As compounds (ESI-MS) Courtesy Jörg Feldmann et al, Aberdeen Univ. Page Page 21 21

22 Separation and Identification of Organo- Arsenic Species ph 5.3 ICP-MS ESI-MS Intensity Peak k1 Peak 2 Peak 3 % [M+H] DMAE Peak 1 (B) ICP-MS m/z 75 y m/z m/z 139 m/z 277 m/z 259 m/z 181 m/z 361 m/z Retention time (s) Chromatograms (above) for ICP-MS measurement of As (mass 75) and ESI- MS of various indicator masses. Mass spectra (ESI-MS) for the 3 peaks show characteristic fragmentation patterns for the identified species DMAE, DMA and (maybe) DMAA % DMA [M+H] + Peak 2 (C ) Peak 3 (D) [M+H] [2M+H] + [2M+H] [2M-H 2 O] m/z % m/z DMAA? m/z Hansen et al. J Anal At. Spectrom, 18, 474 Page Page 22 22

23 Which Elements can be Measured Using ICP- MS? ICP-MS can also measure non-metals used in highly toxic compounds, such as pesticides and chemical warfare agents, provided the backgrounds can be controlled e.g. using GC-ICP-MS Page 23

24 Agilent GC-ICP-MS Interface GC-ICP-MS System used: ICP-MS: Agilent 7500 GC: Agilent 6890 Interface: Agilent G3158A Fully heated and insulated GC transfer line Modified torch with heated injector replaces standard demountable torch Silicosteel transfer line and injector liner for inertness GC effluent injected directly into base of plasma Very high transport efficiency, high plasma temperature (no water vapour/aerosol) and no solvent-based interferences High plasma temperature means elemental response is high, even for poorly ionized elements. Also, elemental response is independent of compound, so compound independent calibration (CIC) is possible Courtesy Raimund Wahlen, LGC Teddington Page 24

25 Pesticide Analysis by GC-ICP-MS Single ion chromatograms for C, P and S (right) and Cl, Br and I (below), extracted from multi-element GC-ICP-MS acquisition Low backgrounds (due to absence of solvent) and good ionization (due to high temperature of dry plasma), leads to excellent signal to background and low LOD Chlorine Ion (34.70 to 35.70): CICCAL3.D Bromine Ion (78.70 to 79.70): CICCAL3.D Iodine Ion ( to ): CICCAL3.D Carbon Ion (11.70 to 12.70): CICCAL3.D Ion (30.70 to 31.70): CICCAL3.D Phosphorus Ion (33.70 to 34.70): CICCAL3.D Sulphur Compound Conc (pg/ul - ppb) Calib Elements Elemental % Dichlobenil 610 Cl ,4,6-TBA 287 Br 72.5 Ethoprop 39 P, S 12.8, 26.4 DBOB 100 Br 35.1 Phorate 210 P, S 11.9, 36.9 PCNB 169 Cl 60.1 Terbufos 745 P, S 10.8, Diazinon 976 P, S 10.2, Malathion 107 P, S 9.37, Dursban 569 Cl, P, S 30.3, 8.82, 9.15 Table (right) shows components, concentrations and elemental l weight % in 1/10 diluted d CIC pesticide id mix. Page 25 Ioxynil (methyl ester) 50 I 66 TPP 158 P 50.3

26 CIC - Sulphur in Pesticide Mix Sulphur elemental response is independent of the compound Sulphur Response Area R 2 = Malathion Ethoprop Phorate Dursban Diazinoni Compound Terbufos Compound Concentration RT S conc S pg/ul (min) (ppb) response Ethoprop Phorate Terbufos Diazinon Malathion Dursban Concentration (ppb) Pesticide compound LoD s typically single ppb or sub-ppb Page 26

27 Chemical Warfare Agent (CWA) Regulation Chemical Weapons Convention of January 1993 Enforcement began April 1997 August 24 th, 2006; Meeting of 180 countries (representing 98% of World Population) which are members of the OPCW. Organization i for the Prohibition i of Chemical Weapons ~70,000 Metric Tons of Chemical Weapons Declared (24/8/2006) ~14,000 Metric Tons Destroyed (24/8/2006) 2006 Budget $96 Million "Determined for the sake of all mankind, to exclude completely the possibility of the use of chemical weapons..." From Doug Richardson, Univ Cincinnati Page 27

28 Chemical Warfare Agent Analysis by ICP-MS Ner ve Agent s G-Type V-Type H 3 C O CH P O C H 3 CH3 F H 3 C CH 3 Soman (GD) H 3 C O CH 3 P O C H CH F 3 Sarin (GB) O O H 3 C P O CH 2 CH 3 C P H 3 S S O CH 3 CH 3 N O O O P CN H 3 C P O N F VX TAbun (GA) Cycl osar in (GF) N Russian VX (RVX) 31 P Selective Detection All these agents contain a P atom, so ICP-MS can be used to identify and quantify the concentration of agent, based on the consistent (compound independent) response for 31 P. Nerve agents mostly decompose in the environment to MPA (via EMPA, IMPA, CMPA ) From Doug Richardson, Univ Cincinnati Page 28

29 CWA Analysis in Natural Samples by LC-ICP-MS Right: Standards Below: Unspiked and spiked Apple Juice Column: Hamilton PRP-X100 Anion Exchange P Respo onse (CPS) P 1 Resp ponse (CPS S) Time (min) Apple Juice + Spike (3ppm) Apple Juice Time (min) Elution Order 1. MPA 2. H 2 PO EPA 4. DMHP 5. PPA 6. EMPA 7. IMPA 8. DEHP 9. IPHEP 10. IBHMP From Doug Richardson, Univ Cincinnati Page 29

30 Forensic Applications of ICP-MS Glass Fragment Analysis Almost any solid fragment collected from a suspect individual or location may be suitable for analysis using laser ablation ICP-MS sample size as small as 50um diameter can be measured routinely Glass samples can be analysed using simple screening scan (qualitative or semi-quantitative) or calibrated against well-characterized reference glasses. NIST 600 series Trace Elements in Glass eg NIST 612 ~ 50ppm Images courtesy of New Wave Research

31 Trace Element Distribution Patterns (sum to 100%) Synthetic ti Glass and Unknown Samples Data courtesy of New Wave Research Page 31

32 Analysis of Bic Black Pen Inks 15 black Bic pen inks different sources Non-ablated Ink analyzed in triplicate 24 elements / 26 isotopes 105 comparisons taken as pairs 86/105 (82%) pairs were discriminated by Pb Of 19 remaining, 17 pairs were discriminated by Co Of 2 remaining, 1 pair was discriminated by Ba Both Zn and Cu have similar discriminating power to Co Only 1 pair (<1%) could not be discriminated Most useful discriminators: Pb, Co, Ba, Cu, Zn Ablated Ink Images courtesy of FBI Academy Page 32

33 Eight Bic Black Pen Inks Unknown #2b is 02 Bic (high W and low Mo) Unknown #8b is 08 Bic (High Pb and low W) Al/Cu Pb W/Mo Mo/Co Zn/Ba Bic Mean 02 Bic Mean 03 Bic Mean 04 Bic Mean 05 Bic Mean 06 Bic Mean 07 Bic Mean 08 Bic Mean Unk (#2b) Unk (#8b) Data courtesy of FBI Academy Page 33

34 Metal Imaging Mass Spectrometry (MIMS) Slice frozen tissue, e.g. brain 10 µm tissue sections LA-ICP-MS Image reconstruction Raster Laser Data acquisition iti Images courtesy of Dominic Hare, UTS Page 34

35 MIMS maps of various elements in brain section of Parkinson ss disease rat model system Intact side of brain EM Lesion side of brain (Parkinson-like) Intensity increases from blue green yellow red 31 P 56 Fe 57 Fe Images courtesy of Dominic Hare, UTS Page Page 35 35

36 Conclusions ICP-MS offers a unique combination of: Wide elemental coverage (almost all elements can be measured) Low limits of detection (typically 10 s ppq for easily ionized elements) Wide dynamic range (from sub-ppt to 1000 s ppm) Very rapid analysis (<3 minutes for full elemental screening, much less for consistent matrices and fewer elements) Freedom from spectral interferences (using collision/reaction cell) Tolerance of a wide range of matrices (due to high plasma temperature) Facility to link to chromatographic and direct solid sampling accessories, to further extend the range of applications Page 36