Analytical Methods For Measuring Lead In Blood
Brief guide to analytical methodsfor measuring lead in blood
WHO Library Cataloguing-in-Publication DataBrief guide to analytical methods for measuring lead in blood.1.Lead - analysis. 2.Blood - analysis. 3.Lead - chemistry . 4.Electrochemical techniques.5.Spectrophotometry, Atomic - methods. 6. Mass spectrometry - methods. I.World Health Organization.ISBN 978 92 4 150213 9(NLM classification: QV 292)This publication was developed in the IOMC context. The contents do not necessarily reflect the viewsor stated policies of individual IOMC Participating Organizations.The Inter-Organization Programme for the Sound Management of Chemicals (IOMC) was establishedin 1995 following recommendations made by the 1992 UN Conference on Environment andDevelopment to strengthen co-operation and increase international co-ordination in the field ofchemical safety. The Participating Organizations are FAO, ILO, UNEP, UNIDO, UNITAR, WHO,World Bank and OECD. UNDP is an observer. The purpose of the IOMC is to promote co-ordinationof the policies and activities pursued by the Participating Organizations, jointly or separately, toachieve the sound management of chemicals in relation to human health and the environment. World Health Organization 2011All rights reserved. Publications of the World Health Organization are available on the WHO web site(www.who.int).The designations employed and the presentation of the material in this publication do not imply the expression ofany opinion whatsoever on the part of the World Health Organization concerning the legal status of any country,territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. Dotted lineson maps represent approximate border lines for which there may not yet be full agreement.The mention of specific companies or of certain manufacturers’ products does not imply that they are endorsed orrecommended by the World Health Organization in preference to others of a similar nature that are notmentioned. Errors and omissions excepted, the names of proprietary products are distinguished by initial capitalletters.All reasonable precautions have been taken by the World Health Organization to verify the information containedin this publication. However, the published material is being distributed without warranty of any kind, eitherexpressed or implied. The responsibility for the interpretation and use of the material lies with the reader. In noevent shall the World Health Organization be liable for damages arising from its use.
ContentsAcknowledgements. iv1. Purpose and scope . 12. Background. 13. Available analytical methods. 23.1 Atomic absorption spectrometry (AAS) . 23.1.1 Flame atomic absorption spectrometry (FAAS) . 43.1.2 Graphite furnace atomic absorption spectrometry (GFAAS). 43.2 Anodic stripping voltammetry (ASV) . 43.2.1 Laboratory ASV devices. 43.2.2 Portable ASV device . 53.3 Inductively coupled plasma mass spectrometry (ICP-MS). 64. Important aspects of laboratory practice. 64.1 Preventing external contamination of samples . 74.2 Quality assurance (QA). 75. Considerations for method selection. 85.1 Purpose and circumstances. 85.2 Availability of operational equipment . 85.3 Ease of use and availability of skilled personnel. 95.4 Analysis costs and availability of financial resources. 95.5 Quality assurance . 106. Scenarios . 106.1 Suspected intoxication . 106.2 Exposure assessment. 116.3 Screening . 116.4 Occupational health . 117. References. 12iii
AcknowledgementsThis document was written by Dr Pascal Haefliger. The following people reviewed andprovided comments on the document, and their contributions are gratefully acknowledged:Dr M. Fathi, Toxicology Laboratory, University Hospital of Geneva, SwitzerlandMr J.M. Jarrett*, Division of Laboratory Sciences, National Center for EnvironmentalHealth, Centers for Disease Control and Prevention, Atlanta, United States of America(USA)Dr I. Naik, Analytical Services, National Health Laboratory Services, National Institute forOccupational Health, Johannesburg, South AfricaDr P. Nisse, l’Unité de Toxicovigilance, Centre Antipoison de Lille, Lille, FranceDr V.V. Pillay, Department of Analytical Toxicology & Forensic DNA Typing, AmritaInstitute of Medical Sciences & Research, Cochin, IndiaMs M. Sucosky*, Healthy Homes and Lead Poisoning Prevention Branch, Centers forDisease Control and Prevention, Atlanta, USA.Dr A. Taylor, Supra-regional Assay Service, Trace Element Laboratory, Centre forClinical Science, University of Surrey, Guildford, England* These individuals served as a technical subject matter reviewers, however, their mention does notindicate their agreement with or endorsement of the document and does not necessarily represent theofficial position of the Centers for Disease Control and Prevention.The document was finalized by Ms Joanna Tempowski, Department of Public Health andEnvironment, World Health Organization (WHO), Geneva, Switzerland. The document wasedited by Ms Marla Sheffer.WHO gratefully acknowledges the financial support of the German Federal Ministry for theEnvironment, Nature Conservation and Nuclear Safety.For further information on this document please contact ipcsmail@who.int.iv
1. Purpose and scopeThis document provides a brief overview of analytical methods commonly used for measuring lead in blood. It is primarily aimed at informing public health personnel and policy-makerswho are not laboratory specialists but who may need to develop plans for population screening and other public health actions related to human exposure to lead. The document listswell-established analytical methods for measuring lead in blood and briefly describes someof their characteristics, including their advantages and disadvantages. It also highlights, forvarious types of applications and scenarios, the considerations that need to be taken intoaccount when selecting an analytical method and when deciding about whether to establisha laboratory service for lead measurement or whether to contract it out. This document doesnot aim to provide an exhaustive description of analytical methods and protocols or to makespecific recommendations regarding methodologies or specific instruments. More exhaustivereviews of this subject are available elsewhere (1), and links to further information andreading are provided in section 7.2. BackgroundLead is a toxic metal whose widespread use has caused extensive environmental contamination and health problems in many parts of the world. Human exposure to lead isestimated to account for 143 000 deaths every year and 0.6% of the global burden ofdisease (2). Lead is a cumulative toxicant that affects multiple body systems, including theneurological, haematological, gastrointestinal, cardiovascular and renal systems. Chronicexposure commonly causes haematological effects, such as anaemia, or neurological disturbances, including headache, irritability, lethargy, convulsions, muscle weakness, ataxia,tremors and paralysis. Acute exposures may cause gastrointestinal disturbances (anorexia,nausea, vomiting, abdominal pain), hepatic and renal damage, hypertension and neurological effects (malaise, drowsiness, encephalopathy) that may lead to convulsions and death.Children are particularly vulnerable to the neurotoxic effects of lead, and even low levels ofexposure can cause serious and, in some cases, irreversible neurological damage. Childhood lead exposure is estimated to contribute to about 600 000 new cases of children withintellectual disabilities every year (3).The clinical diagnosis of lead poisoning can be difficult when there is no clear history ofexposure, because poisoned individuals can be asymptomatic, and signs and symptoms,when they are present, are relatively nonspecific. Laboratory investigations are the onlyreliable way to diagnose lead-exposed individuals and therefore play an essential role in theidentification and management of lead poisoning and in the assessment of occupational andenvironmental lead exposure.Today, laboratories primarily assess lead exposure with whole blood lead measurements.Although a number of other human tissues and fluids, such as hair, teeth, bone and urine,also reflect lead exposure, the concentration of lead in whole blood has gained wideacceptance as the most useful tool for screening and diagnostic testing (1, 4). In very youngchildren, the lead level in whole blood is an indicator mainly of recent exposure, althoughthere can be variable (but not dominant) input to total blood lead concentration from past1
Brief Guide to Analytical Methods for Measuring Lead in Bloodaccumulation of lead in the body. In adults, particularly in lead workers, the past accumulation can be a more prominent contributor to total blood lead concentrations.3. Available analytical methodsA number of laboratory methods are available to determine blood lead concentrations (1, 5–9). The most common are atomic absorption spectrometry (AAS), anodic stripping voltammetry (ASV) and inductively coupled plasma mass spectrometry (ICP-MS). In addition, asimple to use, portable device using ASV technology is available for performing blood leadmeasurements at point of care. These methods differ significantly in their analytical capacities (e.g. limits of detection, accuracy), costs (e.g. purchase and maintenance costs, laboratory infrastructure required, reagents and supplies) and technical requirements (e.g. samplepreparation, calibration, skilled personnel). These factors, taken in conjunction with the setting and resources of the laboratory, will influence the decision about the choice of method.The required limit of detection is an important consideration. In many countries, there hasbeen a successive reduction in the blood lead concentration considered to be of clinical concern. This reflects the growing body of evidence suggesting that there may be no thresholdconcentration of lead in the body below which there are no adverse health effects (10). Inaddition, public health measures in a number of countries have succeeded in reducing themean blood concentration in populations. An example is the USA, where the geometricmean blood lead concentration in the population has decreased from 15–17 µg/dl in the mid1970s (11) to the current value of below 2 μg/dl (12). These two factors have increasedinterest in measuring ever-lower blood lead concentrations and created a need for analyticalmethods that can perform at low levels of detection. In situations where population or subpopulation blood lead concentrations are still elevated, some older technologies with higherlevels of detection may still be applicable.Further discussion about different analytical methods is provided in the sections below andsummarized in Table 1.3.1 Atomic absorption spectrometry (AAS)AAS is based on the fact that free atoms absorb light at wavelengths characteristic of theelement of interest. The amount of light absorbed can be correlated in a linear fashion to theconcentration of the analyte in the sample. To conduct an AAS measurement, the leadcontaining sample must first be processed by the instrument so as to generate ground-stateatoms as a vapour within the light path of the instrument. This process, called atomization,can be done using either a flame (flame atomic absorption spectrometry, or FAAS) or anelectrothermal source, most often a graphite furnace (graphite furnace atomic absorptionspectrometry, or GFAAS). Although FAAS and GFAAS have similar detection principles,they differ greatly in their applicability to direct measurement of lead in blood (e.g. limits ofdetection, sample size, sample preparation).2
Brief Guide to Analytical Methods for Measuring Lead in BloodTable 1. Overview of analytical methods for blood lead measurementMethodStrengthsLimitationsFlame atomicabsorptionspectrometry (FAAS) Requires only basic laboratoryexpertise Relatively high detection limit( 10 µg/dl) Rapid analysis Time needed for sampledigestion/preconcentration ifnot using Delves cup Small sample size using Delvescup (50–100 µl) Low purchase and running costs Relatively few interferencesGraphite furnaceatomic absorptionspectrometry(GFAAS) Large sample size needed fornebulization methods Robust interface Should not be left to rununattended Good detection limit ( 1–2 µg/dl) Longer analysis time Small sample size Requires some laboratoryexpertise (more than FAAS) Moderate purchase and runningcosts Some multielement capacity Greater potential spectralinterference than with FAAS Relatively few interferences(although more than with FAAS) Widely used, available frommultiple vendorsLaboratory anodicstripping voltammetry(ASV) Good detection limit (2-3 µg/dl) Low purchase and running costs Requires some laboratoryexpertise (similar to GFAAS) Rapid Sample pretreatment needed Small sample size ( 100 µl) Some factors might affectmeasurement (e.g. presence ofcopper) Relative simplicity of equipment Becoming less availablePortable ASV Portable; measurement at point ofcare possible Not as accurate as othermethods Simple to use; does not requireskilled laboratory personnel Can determine levels only up to65 µg/dl Very low purchase and runningcosts Levels above 8 µg/dl should beconfirmed by a laboratorymethod Reasonably good detection limit fora portable device (3.3 µg/dl) RapidInductively coupledplasma massspectrometry (ICPMS) Excellent method detection limit( 0.1 µg/dl) High purchase and runningcosts Rapid Highly skilled laboratoryoperator required Small sample size (50–100 µl) Relatively few, well-understood,spectral interferences Isotopic measurements possible Economic if very large number ofsamples Multielement capability3
Brief Guide to Analytical Methods for Measuring Lead in Blood3.1.1 Flame atomic absorption spectrometry (FAAS) (5, 13)FAAS uses an acetylene–air or a nitrous oxide–acetylene–air laminar flame to atomize leadat temperatures in the order of 2000–3000 C, depending on the gas mix. The limit of detection of FAAS for blood lead depends on sample preparation and the method used. Delvescup methods, for example, enable the use of 50–100 µl sample sizes with a limit of detectionin the order of 10–30 µg/dl. By comparison, using nebulization methods, the limit of detectionis around 100 µg/dl, and larger samples are needed. Even the lowest achievable limit ofdetection is too high for FAAS to be useful for screening in populations with low backgroundblood lead concentrations.FAAS devices can be fitted with an autosampler, which enables large numbers of samples tobe processed. Because they use a flammable gas, however, FAAS devices should not beleft to run unattended. Owing to its relative simplicity of use, rapidity, relative freedom frominterferences and moderate cost, FAAS has been used for decades and is still in routineoperation in many parts of the world. In many countries, however, FAAS has largely beensuperseded by GFAAS, which can determine much lower blood lead concentrations.3.1.2 Graphite furnace atomic absorption spectrometry (GFAAS)GFAAS uses an electrically heated graphite tube to vaporize and atomize the analyte attemperatures up to 3000 C prior to its detection. Sample volumes of 10–50 µl can beanalysed. Because the entire sample is atomized within a small volume, a dense atom population is produced. This technique is therefore very sensitive. Methods have been developedthat can measure lead concentrations down to below 0.1 µg/dl (6, 14); however, in routineuse, the limit of detection is in the order of 1–2 µg/dl. GFAAS is currently one of the mostcommonly used methods for determining lead concentrations in blood. GFAAS is subject togreater potential interference than FAAS. This potential for interference has been reduced byimproved instrumentation design and by the application of various matrix modifiers. GFAAS,however, requires trained laboratory personnel to be set up and operated accurately.Modern GFAAS instrumentation is reliable, accurate and precise. GFAAS devices are usually equipped with an autosampler, which allows for large numbers of samples to be processed and better precision. The fact that GFAAS uses inert gas means that it can safely beoperated unattended. Several manufacturers market GFAAS instruments that are readilyconfigured for blood lead testing. GFAAS can be used for limited sequential analysis ofmultiple elements (e.g. lead and cadmium) in a single sample. It can be set up to measure awide range of elements as a single element per sample.3.2 Anodic stripping voltammetry (ASV)3.2.1 Laboratory ASV devicesTo conduct an ASV measurement, a reference electrode and a thin-film mercury graphiteelectrode are placed in the blood sample. A negative potential is then applied to the mercuryelectrode for several seconds, which causes lead and other cations present in the sample toconcentrate on the surface of the negatively charged mercury electrode. The direction of thepotential is then reversed to give an increasingly larger potential over several minutes. Asthe voltage reaches a specific and characteristic voltage for lead, all such ions are released(stripped) from the electrode, thereby producing a current that can be measured. The current4
Brief Guide to Analytical Methods for Measuring Lead in Bloodproduced is proportional to the number of lead ions released and can be compared withcalibration solutions to determine the lead concentration in the sample. This analyticaltechnique requires that lead be decomplexed and available as the free Pb2 aqueous cationand therefore involves sample preparation.Although ASV can be used to measure a number of elements, it is mainly used
Brief Guide to Analytical Methods for Measuring Lead in Blood accumulation of lead in the body. In adults, particularly in lead workers, the past accumu-lation can be a more prominent contributor to total blood lead concent
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