Well-being through work COPHES301111 · Well-being through work ... • Sample preparation •...

© Finnish Institute of Occupational Health – www.ttl.fi30.11.11

Well-being through work

© Finnish Institute of Occupational Health – www.ttl.fi30.11.11 2Mirja Kiilunen

Analysis of Cd and creatinine in urine Workshop on Quality of Analytical Data in HBM30.11.11, Brussels, Belgium Organized by WP 3 of COPHESMirja Kiilunen


Outline

• Problems with sample, samples storage, shipping etc.

• Choice of method• AAS• ICP-MS

• Creatinine measurements• Modification of Jaffe’s


Problems for sample

• Sample collection• Dilute samples• Wrong sampling time

• Sample Storage• Shipping

• Sample preparation

• Analysis

• Interpretation


Sample Transport and Storage

• Losses• Absorption• Precipitation• Degradation

• Stability• Storage temperature• Storage time• Growth - preservative ?

• Shipment


Urinary Cadmium - sampling

• Special-washed laboratory ware is required for successful specimen collection

• Also clean collection cup • After shower or washing hands and the urethral

orifice and change to street clothes • No preservatives

• Sampling time is not critical for cadmium• Dilute samples

• creatinine in urine of adult working Finns is 13 mmol/l urine equate relative density 1,021


Guideline for sampling U-Cd

Exposure Cadmium and inorganic cadmium compounds

Specimen 20 ml of urine. Special-washed containers have to be used, and no preservatives may be added. If the urine sample is voided to a disposable vessel, it has to be transferred immediately to the special-washed sample container. The laboratories of the Institute of Occupational Health provide special-washed sample containers.

Sampling time Specimen can be collected at any time of the day

Storage and transport The specimen is stored in a refrigerator and sent so that it reaches the laboratory before the week-end. Specimens collected toward the end of the week are preferably kept in the refrigerator over the week end and sent on Monday.

Sources of error Specimen is prone to contamination. Dust from the work place air, or skin or clothing of the workermay contaminate the specimen. The specimen is collected after the worker has showered (or washed hands and the urethral orifice) and changed to street clothes.

Note The concentration of antimony, beryllium, chromium, cobalt, lead, manganese, molybdenum, nickel, selenium, thallium and vanadium may be determined from the same 20-ml specimen if the sampling time is after shift.

Reference limit for non-exposed

Non-smokers 5 nmol/l.Smokers 10 nmol/l.

Biomonitoring action limit

40 nmol/l.The European Union has classified several cadmium compounds as carcinogenic, mutagenic or hazardous to reproduction (Cat. 2 or 3; R45, R63, and R68, H340, H341, H350, H360FD, H351fd). The Finnish Government Decree 1335/2004 stipulates that exposure to chemicals in these categories is not acceptable during pregnancy, i.e., the urine cadmium concentration must not exceed the reference limit for non-exposed 5 nmol/l (non-smokers) or 10 nmol/l (smokers).

www.ttl.fi/biomonitoring


Analysis of cadmium in urine

• Colorimetric methods Diphenylthiocarbazoneand dithizone – CHCl3

• a suitable method should measure 0-20 μg Cd in 50 ml urine and be sensitive to 0.1 μg Cd. ~ 400 μg/l ~3.6 μmol/l

• Great problems from other metals Cu++ >30 μg prevent detection of Cd. Pre-extraction with dithizone – CHCl3 fromacidified solution and Cd from basic solution afterwards.

• Dithizone was also photolabile – prevention NH4 , KCN• Two prewashings of dithizone solutions• Acid digest of urine on sand bath followed by three different

kind of extraction processes• Colorimetric measurement at 506 nm• 12 urine samples + 2 blanks at a time• Lowest concentrations detected 9 μg/l ~85 nmol/l

SMITH et al. Biochem J. 1955 Dec;61(4):698–701 .


Analysis of cadmium in urine

Atomic absorption flame technique• metals extracted as iodide complexes with tri-

n-octylamine in n-butyl acetate• without elaborate pretreatment, direct

nebulization into an air/ acetylene flame• Detection limits for cadmiumin urine

0.008×10 6 mol/l ~ 0,8 nmol/l• No information of control samples

Flanjak, J, Hodda, A. (1985) Analytica Chimnica ActaVol 172:313-316


Different derivate flame techniques• Delves cup and other derivate techniques

Urine samples 100 ml digested on an electric hot-plate with HNO3, HCIO4 for 1 hour, residue dissolved into HNO3

Concentrated on a water-cooled stainless steel atom trapping tubeDL 0.02 μg/l~0.2 nmol/l

The absorption signals of conventional AT-FAAS (l) and D-AT-FAAS (II)

Sun Han-Wen et al. (1997) Spectrochimica Acta Part B 52 727-734


Flameless Atomic absortion

• ‘Reference method’ for urinary cadmium measurements

• Ammoniumpyrrolidine hexamethylenedithiocarbamate (APDC)/MIBK + FAAS

• DL 0,2 μg/l ~ 2 nmol/l

Angerer in Analyses of Hazardous Substances in Biological Materials,Vol 2, VCH, 1988, 85-96


Flameless AAS

• Standard addition method• Dilution 1:3 with nitric acid• Modifier Ammonium oxalate

• Detection limit 0.01 μg/l ~ 0.1 nmol/l

Kiilunen, M., Aitio, A. (1990) International symposium on trace elements in health and disease., 5.-8.6. Espoo, Finland


InductivelyCoupled Plasma Atomic Emission Spectrometry ICP-AES (OES)

• 1,5-bis(di-2-pyridylmethylene)thiocarbonohydrazide extraction into isobutyl methyl ketone

• Critical effects: pH, concentration of extractant, shaking time and ionic strength

• Detection limit 0.1 μg/l ~0,9 nmol/l• Linear area 0.2 -140 μg/l, CV 2,9 % for 2 μg/l

Espinosa Almendro et al, ANALYST, 1992, VOL. 117, 1749-51


ICP-AES

• Purification of samples with activated charcoal under acidic conditions

• preconcentration APDC /MIBK• the resulting residue is finally digested under acid

oxidant conditions • detection limit (below 10 μg/l)• this procedure can be applied conveniently for

toxicological diagnostic purposes

• Also phase extraction methods are used• Internal standards to ensure reproducibility

López-Artiquez et al. (1993) J Anal Toxicol 17(1):18-22.


Validation of the method

Selectivity and specificity• AAS the emission spectrum – resonance line

• a definite frequency or definite frequencies; second-degree line

• ICP-MS Mass-to-charge ratio m/z

• ICP-OES >< ICP-MS 48Cd Detection limits• 5 μg/l solution ICP-OES• 0.0005 μg/l solution ICP-MS• 2.5 μg/l solid ICP-OES• 0.01 μg/l solid ICP-MS

(theoretical best case scenarios)



• range of measurement and linearity

• Important, if wide concentration range

• ICP – huge, AAS - limited

• In practise range of standards - dilutions



Sensitivity

AAS• the Lambert–Beer law

• A= * l* c• = molar absortivity, l = length of cuvette

ICP-MS• c/s = (BLANK + a + b*conc)*[I/S Ratio].


AAS >< ICP-MS

0 5 10 15 20-2

0

2

4

6

8

10

12

14

16

18

ICP

-MS

μg/

l

AAS μg/l

Y = 0.068 + 0.934 * XR = 0.995 N=50 P<0.0001



limit of detection LOD • detection limit, is the lowest concentration level

that can be determined to be statistically different from a blank (99% confidence)

• X ± 3*sdlimit of quantitation LOQ • lower limit of quantitation (LOQ), is the level

above which quantitative results may be obtained with a specified degree of confidence.

• X ± 6*sd



• Method interferences• AAS

• Matrix effects, chemical effects, spectral interferencesminor physical and ionisation (GAAS)

• ICP-MS• Argon, nitrogen, oxygen, chlorine and hydrogen, and

their constituent compounds• Spectral disturbances caused by occultation• Spectral errors due to mono-and polyatomisista ions -

isobaric disturbances• Matrix effects



Precision• a series of repeated measurements of the same

parameter (External quality control or spiked samples)

Repeatability• a series of repeated preparations and

measurements of the same parameter within a sample

• Different concentration levels on range of measurement

• measurement uncertainty


Plasmas

• A sample solution is introduced into core of inductively coupled argon plasma (ICP)

• 8000 °C

• OES: All elements thermally excited and emit light at their characteristic wavelenghtsthrought diffraction grating amplified signal

• MS: plasma generates ions mass analyzermass to charge ratio


Inductively Coupled Plasma Spectrometry techniques

Strengths Limitations

Up to 70 elements can be determinatedsimultaneously

The emission spectra are complex and inter-element inferences are possible – wavelength very close to that another element.

The useful working range is over several orders of magnitude

MS. Common matrix elements and other molecular species can interfere with the measurement of some elements. Doubly charged or molecular ionic species can create difficulties in quantifications.

Instrument suitable to automation, thus enhancing accuracy, precision and throughput.

The sample to be analyzed must be digested or at least diluted prior to analysis in order to dissolve the elements of interest.


Cadmium and Tin interferences

111Cd

112Cd -0.04000 * 118Sn

114Cd -0.02700 * 118Sn


Standard curves


Do not use Cd 108 and 110


Creatinine

• Creatinine is a waste product of muscle creatine

• The amount of creatinine produced is related to body weight

• It does not vary greatly from day to day

• Within subject variation4.3 %

Creapure®


Creatinine and diet

Hoogwerf BJ, Lame DC, Greene, E (1986) Am J Clil Nut 43: 350-360.


Jaffe’s reaction

• Creatinine + Picric acid Alkaline pH Yellow complex• absorbs at 510 nm

• Any compound that reacts with picrate in alkali conditions e.q. protein, glucose

Jaffe M. Ueber den Niederschlag welchen Pikrinsäure in normalen Harnerzeugt und ueber eine neue Reaktion des Kreatinins. Z Physiol Chem 1886;10:391-400


Modifications

• Deproteinisation to improve specificity• Absorption on aluminium silicates to remove

interferents followed by elution into alkaline picrate

• At a neutral pH only the interferents reacts

• Automatisation started on 1960’s• Continous flow technology• Removing protein, bilirubin, not glucose.


Entsymatic reactions

At 670 nm

Interferences: creatine, proline, dobutamin, lidocaine


VITROS is a trademark of Ortho-Clinical Diagnostics, Inc.© Ortho-Clinical Diagnostics, Inc., 2007.

Preservatives – no interferences

Effect < 2% on creatinine results• Thymol• Toluene• Boric acid• Glacial acetic acid (0.5 to 1.0 v/v)• 12N HCl• NH4OH• Bromide• Iodide• 5% NaOH


Problems with kits

Creatinine (urinary) Assay Kit Item No. 500701©06/22/2011, Cayman Chemical Company

Intra-assay CV = 2.7% (n = 84)Inter-assay CV = 3% (n = 5)


Sample kit Vitros


HPLC methods

• Diluted urine + acetonitrile containing phenacetin(internal standard) centrifugated supernate directly into the chromatograph

• Isocratical liquid chromatography• mobile phase of acetonitrile and aqueous KH2PO4

Sample 1 Sample 2 Sample 3

X mg/l 407 814 1157

SD mg/l 5.4 21.9 21.6

CV% 1.3 2.7 1.9

Between-day (n=6, each run on 12 days)

Okuda, T, Ole, T, Nishida, M (1983) CLIN.CHEM. 29/5,851-853


Creatinine in urine –microtitreplates – Jaffé method 492 nm

Concentration level Control Intra-assay repeatability(N=10)

1.6% or 1.6 % 0.79g/l or 1.71 g/l Duotrol®Standard deviation (rel.) 2.1% or 2.5 % 0.72 g/l or 1.40 g/l Quantimetrix®

3.6% or 3.6 % 0.79g/l or 1.71 g/l Duotrol®Confidence interval 4.7 % or 5.7 % 0.72 g/l or 1.40 g/l Quantimetrix®

Inter-day repeatability (N=6) 1.5% or 1.4 % 0.80g/l or 1.72 g/l Duotrol®Standard deviation (rel.) 1.4% or 2.2 % 0.72 g/l or 1.40 g/l Quantimetrix®

3.9% or 3.6 % 0.80g/l or 1.72 g/l Duotrol®Confidence interval 3.6 % or 5.7 % 0.72 g/l or 1.40 g/l Quantimetrix®

Accuracy (N=8) 107.9 % or 102.6% 0.72g/l or 1.66 g/l* Duotrol® Recovery rate 93.7 % or 84.6 % 0.78 g/l or 1.63 g/l* Quantimetrix®

Detection limit <0.125 g creatinine per litre urine *theoretical values

Blaszkewicz and Liesenhoff-Henze MAK-Collection for Occupational Health and Safety 2010


Creatinine – Jaffe’s method

Principle of the method• Creatinine present reacts with alkaline picrate

resulting in the formation of a red colour

• the intensity is measured at 505nm/green filter• protein interference elimination: sodium lauryl

sulphate• A second absorbance reading after acidifying with

30% acetic acid corrects for non-specific chromogens in the samples.

Fossati,P, Ponti,M, Passoni,G, Tarenghi, G, Meizi d’Eril, GV, Prencipe, L (1994) CLIN. CHEM. 40/1, 130-137


Enzymatic method – with iminohydralase

measured at 340 nm the consumption of NADPH (reduced Nicotinamide Adenine Dinucleotide Phosphate)


• Hydrolysation by creatinine iminohydrolaseto ammonia and N-methylhydantoin.

• Consumption of NADPH, is proportional to the amount of creatinine in the sample.

• The first step eliminates background absorbance

• The second step starts creatinine measurement.


Creatinine urine comparison


Present methodinstrument

ComparisonmethodInstrument

n slope X Y Bias % r

TechniconRA-XT

Jaffékinetic/RA-XT

60 1.001 13.7 13.9 0.2(1.4) 0.998

TechniconRA-XT

EntsymaticPAP/Cobas Fara

87 1.015 13.8 14.2 0.4(2.9) 0.996

Hitachi 717 Fuller’searth

60 1.024 4.6 4.9 0.3(6.5) 0.998


Three different enzymatic methods

Badiou, S, Dupuy, A.M. Descomps, B, Cristol, JP (2003) J Clinl Lab Anal 17:235–240

© Finnish Institute of Occupational Health – www.ttl.fi30.11.1112.12.2011 41Mirja Kiilunen

Comparison – different methods instruments


Enzymatic creatinineinterferences and limitations

• Entzymatic reactions are more specific for creatinine

• Many interferences depending the concentrations


Ion-exchange methods

• Different commercial batches of Fuller's earth (cation exchange )

• Incomplete adsorption of creatinine• Depends on pH, temperature, concentration of

cations as Ca2+ and Na+

• Weakly acidic suspensions change their ability to adsorb creatinine

• Synthetic strong acidic cation exchanger more selective to creatinine

Egense, Koch Willems (1990) Scand J Clin Lab Invest. 50(6):687-92.


GC-IDMS – methodsisotope dilution mass spectrometry

• Standard Reference Material 909 of the National Institute of Standards and Technology

• Excellent specificity and relative SD <0.3%• Creatinine is derivatized before GC analysis

because of its polarity• A cation-exchange clean-up step before GC

analysis to remove creatine

• HPLC with IDMS• a simple protein precipitation without

derivatization bias <0.2% and an expanded uncertainty <0.3% (k = 2)

•Stockl and Reinauer (1993) Clinical Chemistry 39: 993-1000.


Reference materials

• NIST SRM 967 two levels 66 and 347 μmol/l

Manufacturer Reagent (Product Number)

Abbott Diagnostics Creatinine (7D64)

Abbott Diagnostics Creatinine (7D64)

Abbott Diagnostics Creatinine, Next Generation (3L81)

Beckman Coulter, Inc. CREA (442760)

Beckman Coulter, Inc. CREA (442760)

Beckman Coulter, Inc. Creatinine (443340)

Beckman Coulter, Inc. CREm (472525)

Dade Behring, Inc. Dimension® CREA Flex (DF33A)

Olympus Diagnostics Systems Creatinine (OSR6178)

Olympus Diagnostics Systems Creatinine (OSR6178)

Ortho Clinical Diagnostics CREA Slides (814 1947)

Roche Diagnostic Corporation CREA plus (11775642)

Roche Diagnostic Corporation CREA/RB (12217333)

Siemens Diagnostics Creatinine_2 (B01-4126-01)

Siemens Diagnostics Creatinine Enzymatic (B01-4127-01)


QC round LABQUALITY


U-Creat mmol/l

Method Mean SD CV% Number of results

Photometric, Jaffe 12.6 0.8 6.2 53

Photometric enzymatic 12.4 0.6 4.7 48

Vitros 11.1 0.8 7.3 8

Method unknown 12.6 0.1 1.1 2

All together 12.4 0.8 6.3 111


Thank You!

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