Routine clinical determination of lead, arsenic, cadmium, and thallium in urine and whole blood by inductively coupled plasma mass spectrometry

SPECTROCHIMICA ACTA PARTB

ELSEVIER

Spectrochimica Acta Part B 51 (1996) 13-25

Routine clinical determination of lead, arsenic, cadmium, and thallium in urine and whole blood by inductively coupled plasma mass spectrometry D a v i d E. N i x o n , T h o m a s

P. M o y e r *

Metals Laboratory, Division of Clinical Biochemistry and Immunology, Mayo Clinic, Rochester, MN 55905, USA

Received 30 January 1995; accepted 31 May 1995

Abstract

For the measurement of As, Cd, Pb, and T1 in urine or whole blood, judicious choices of internal standard elements for matrix correction and the development of a refined isobaric arsenic correction are necessary to produce accurate ICP-MS results. Ga and Rh are chosen as internal standards for As and Cd respectively. Bi is better for the correction of Pb and T1 than Re. An empirically derived equation relating the measurement of ~6035C1 to the 4°Ar35C1 contribution to the arsenic signal at mass 75 is refined by measuring the responses at mass 51 and 75 for urines with added hydrochloric acid. Overall, ICP-MS results for blood and urine are within 6% of Zeeman GFAAS results for patient samples. For surveys, the overall average of ICP-MS results is within 3% of target. Keywords: Arsenic; Blood; Cadmium; Inductively coupled plasma mass spectrometry; Lead, Thallium; Urine;

Whole blood

Dedication

This paper is dedicated to the memory of Richard N. Kniseley. It has been said that sentimentality is the greatest enemy of truth. While these occasions often are sentimental, in truth, Dick Kniseley was a mentor and friend to all who studied and worked in the Spectrochemistry Group at Ames Laboratory. For all of us former graduate students that friendship lasted many years beyond our time at Iowa State University. Dick and Martha always

* Corresponding author.

made time to visit. News of Dick's death was sad indeed, but, he will always be remembered for his acquired knowledge, his willingness to share it, his faithful service to Ames Laboratory and the community, and his unending friendship.

1. I n t r o d u c t i o n

Lead, arsenic, cadmium, and thallium have the distinction of being ubiquitous in our environment because of careless industrial, argicultural, and personal use. These elements and compounds of these elements are toxic to humans and animals in small

0584-8547/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved SSDI 0584-8547(95)01372-5

14

D.E. Nixon, T.P. Moyer/Spectrochimica Acta Part B 51 (1996) 13-25

doses [1-4]. The order of the elements listed above represents not only the decreasing magnitude of universal human exposure but also the decreasing magnitude of society's concern about exposure. Lead seems to be of great concern, primarily because of gasoline combustion, but thallium rarely receives press except when murderous intentions are involved [4]. All of the elements listed above can be involved in chronic low dose exposure due to environmental contamination and the resulting inhalation of aerosol pollution or contaminated food consumption; however, arsenic and thallium are notorious because of their involvement in acute exposure due to ingestion by either accidental, suicidal, or homicidal intent [1-5]. While the blood-urine-tissue partition, target organ, and half-lives of each element in blood and urine differ greatly and depend on whether the exposure was chronic or acute, all of these elements are both nephrotoxic and neurotoxic [1-7]. A common mode of action for all of these elements is enzyme inhibition by binding with sulfhydryl (-SH) groups, resulting in peripheral neurological dysfunction and damage to renal tubules. In acute exposure, the outward physical symptoms are strikingly similar. The only pathognomonic symptom is excess element concentration in the blood and urine. In acute exposure, and also in long term chronic exposure, it is imperative that rapid and accurate analyses be performed in order that rapid diagnosis and treatment be accomplished. In general terms, the rapid analyses of both whole blood and urine is advantageous because moderate to high concentrations of an element in urine would indicate chronic long term exposure or a delay in medical treatment after exposure. These elements are primarily excreted in the urine [2,3]. If significant concentrations are found in whole blood, acute exposure is indicated and immediate action should be taken. This is particularly true for As, Cd, and T1 because they are cleared very rapidly from whole blood. These elements found in whole blood indicate significant exposure at the time the blood sample was drawn [2]. Whole blood Pb is the best indicator of exposure even though clearance from blood is significantly longer because of binding in the red cell [2]. The rapid simultaneous determi-

nation of all of these elements in both blood and urine is advantageous because not only is the toxic element identified quickly but the acute-chronic question can be answered quickly. Over the last l 5 years in this clinical laboratory we have found that graphite furnace atomic absorption spectrophotometry was the most convenient method for trace element analysis of urine, whole blood, and serum. This technique is sensitive at physiologically normal concentrations and little sample preparation is required. Appropriate methods for arsenic [7], lead [8-10], cadmium [9,10], and thallium [11] have been published. The only potential drawback of this technique for clinical analyses is the time and effort needed to complete the determination of arsenic, lead, cadmium, and thallium. In our laboratory two Zeeman-effect graphite furnaces and about one and a half work shifts are needed to analyze an average of 60 specimens per day for all of these elements. Inductively coupled plasma-mass spectrometry potentially offers a solution to the problems of time and manpower needed to complete these toxic metal analyses. Sensitivities are better than graphite furnace. A significant body of data already exists on the application of this technique to biomedical analyses including urine [12-15] and blood or serum [12,14,16,17] specimens. It is significant that a heavy metal screen panel has already been proposed [18] but, to our knowledge the analyses have not been performed with simple aqueous calibrating standards followed by direct analysis of diluted urine or blood. It is evident from the literature and from our preliminary trials that two problems must be solved for accurate and precise analyses to be performed. Firstly, adequate isobaric correction for the 4°Ar35C1 interference at mass 75 must be made so that arsenic can be accurately analyzed. Secondly, appropriate internal standards must be chosen that allow correction of the so called matrix effect or ion suppression, and instrument drift. 1.1. Isobaric interferences

No significant isobaric interferences exist for thallium in serum and urine. For lead at mass 204 mercury interferes with an abundance of 6.85%.

D.E. Nixon, T.P. Moyer/Spectrochimica Acta Part B 51 (1996) 13-25

For cadmium at masses 114 and 112 the only significant interference is tin with abundances of 0.96% and 0.66% respectively. Measurement of tin at mass 118 can be used to correct for any concentration of tin contamination present in the urine. While molybdenum may interfere as MoO + at masses encompassing Cd, with normal physiological concentrations of less than 1 Ixg 1-~ for blood and less than 50 Ixg 1-1 for urine and actual MoO + less than 3% of the nominal Mo concentration, essentially no interference would be expected. There remains only the interference of 4°Ar35C! on arsenic at mass 75 to deal with. The subject of isobaric interferences in inductively coupled mass spectrometry was exhaustively reviewed recently [19]. From this review it appears that three approaches have been used to minimize or eliminate the 4°Ar35C1 interference on arsenic. One has been the use of hydride generation or chromatography to separate the chloride from the arsenic. For our purposes this choice is not satisfactory because three other elements must be determined simultaneously with the arsenic. A second approach has been the addition of a molecular gas such as nitrogen to reduce the formation of 4°Ar35C1. While this interference is reduced, ionization temperatures are also reduced. The net effects on sensitivities for the other analytes are unknown. The third approach is isotopic correction using either an empirically derived relationship of a measured isotope such as ]6035Cl with 4°Ar35Cl [20] or using the classical 4°Ar37C1/82Se/83Kr correction based on known isotope abundances. In this paper the classic 4°Ar37Cl/82Se/83Kr correction and other empirical corrections, including the 16035C1 briefly examined previously [20], are evaluated with real urine specimens. 1.2. Internal standard Internal standardization in flame and plasma emission spectroscopy has generated a wide assortment of rules and opinions about proper internal element selection [21-23]. In like fashion, the use of internal standardization in ICP-MS for the correction of mass spectral interferences has also generated a wide variety of opinions on internal standard element selection and corrective action

15

[15,24-26]. From these papers, it is evident that the most effective internal standard elements for ICP-MS are those that behave almost identically to the analyte. That is, nebulization-aerosol mass transport, ionization energy, and mass number must be nearly identical to the analyte. Just as in the case of ICP-AES where ionization energy is not an infallible guide to the choice of internal standard [21], it appears the same in ICP-MS [25 26]. In a thorough study of ICP-MS internal standardization Thompson and Houk [25] have suggested that matrix interference is affected by plasma operating conditions, ionization energy, sample matrix, and mass of the internal standard relative to the analyte, among other factors. For urine, where salt concentrations such as NaC1 can vary from 50 to more than 250 mM, it can be hypothesized that all of the conditions outlined by Thompson and Houk might change from urine to urine. It would seem logical then that the analyte and internal standard element must experience nearly identical conditions from the bulk urine in the sample cup to their arrival at the mass detector. Instead of internal standardization Wiederin et al. [15] have opted for on line standard addition as a matrix compensation technique for urine analyses. Certainly, no internal standard is needed and the results for Cd and Pb are nearly identical to the certified concentrations in SRM 2670 Freeze Dried Urine. This technique of standard addition, even though performed on line, is simply too tedious and slow for routine clinical analyses. In our first attempt at heavy metal analyses with an ICP-MS, a urine based multielement multiconcentration set of calibration standards was created that contained Y, Rh, and Re as internal standards. It was thought that matrix calibration standards plus internal standardization would be sufficient to correct for variations in aerosol mass trnasport, ionization interferences, and variations in ion transport through the quadrapole from urine to urine. The correlations of As, Cd, and Pb concentrations in real urines and National Institute of Standards and Technology Standard Reference Material (NIST-SRM 2670) between the ICP-MS and Zeeman graphite furnace atomic absorption were marginal at best [27]. Results for Pb and As were parti-

16

D.E. Nixon, T.P. Moyer/Spectrochimica Acta Part B 51 (1996) 13-25

cularly disappointing. Arsenic results were all higher than the Zeeman graphite results by about 30%. Results for urine lead were lower than the Zeeman graphite furnace results by about 20%. The partial success of our initial I C P - M S evaluation suggested that a more thorough study of internal standardization and isobaric correction were needed even with matrix standards calibration. The focus of this work then, is not only on isobaric correction for arsenic, but also on internal standardization for the direct determination of As, Cd, Pb, and T1 in urine and blood. It was the goal of this work to show that simple aqueous acidic standards and simple dilution of all urine and blood specimens with appropriate diluent-internal standard solution would suffice to produce rapid accurate results.

2. Experimental 2.1. I C P - M S facility

All I C P - M S experiments were performed on a Perkin-Elmer Sciex Elan 5000A instrument equipped with an As-90 autosampler. Platinum sampler and skimmer cones were used for all experiments. Typical operating parameters are listed in Table 1. The mass flow controller for nebulizer Table 1 Instrument parameters and elemental equations used in the heavy metal screen analysis Instrument Dwell time/ms Sweeps per reading Resolution Power/W Plasma gas/1 min-~ Intermediate gas/1 min ~ Nebulizer gas/1 min-]

Perkin-Elmer Elan 5000A 150 25 Normal 1000 15 1 0.975

Element equations Arsenic =/As 75 - 0.07898 × loci sl Lead = lpb 2o8 + Ipb204+ lpb 206 + /Pb 207 Cadmium = lc~ ~4 - 0.02747 × Is. 1IS+ IC~ Ill + IC~ tl2 -- 0.03995 × IS..8 Thallium = l~n205 + lTi 203

gas flow, aerosol injector orifice position relative to the sampling cone, and mass spectrometer lens settings were adjusted on a weekly basis. Counts per second were maximized for Rh, Mg, and Pb by sequentially adjusting the above parameters while a solution containing 10 ixg 1-1 of each analyte in 1% HNO3 was nebulized into the plasma. Typical counts for Rh after optimization were 5 × 10+6 (counts × L)/(s × mg). Peak widths were also adjusted at the same time to maintain a 0.7 ___0.1 u peak width for J°3Rh and a maximum difference in peak width between 24Mg and 2°8pb of 0.02 u. 2.2. Z e e m a n G F A A S facility

All Zeeman GFAAS analyses were performed on a Perkin-Elmer Model 5100 ZGFAAS equipped with an As-60 autosampler, HGA-600 graphite furnace, and Model 7700 Professional computer. The Zeeman GFAAS was used as the reference method because routine determination of As, Cd, Pb, and T1 are performed daily for these analytes in urine and blood using methods already described in the literature. It is not the purpose of this publication to completely describe the Zeeman GFAAS methods and sample preparation therefore only comparative results for Zeeman GFAAS versus I C P - M S are listed. The quantitative recovery of these analytes by Zeeman GFAAS has been routinely verified by analyses of survey samples such as those from the Centre de Toxicologie du Quebec, Centers for Disease Control and Prevention and NIST SRM materials. For the purposes of this publication we consider the Zeeman GFAAS technique to yield the correct answers unless otherwise noted. Briefly, urine samples, controls, and standards are diluted with aqueous nitric acid and placed in the autosampler cups. Chemical modifiers, such as palladium plus potassium persulfate for the analysis of arsenic [28], are added to the graphite platform after the diluted sample is deposited. The sample plus chemical modifier is dried, charred, then atomized at the appropriate temperature. Argon is used as the flush gas in all analyses. Multielement multiconcentration urine or aqueous based standards are used for instrument response

D.E. Nixon, T.P. Moyer/Spectrochimica Acta Part B 51 (1996) 13-25

calibration. Quality control urine, whole blood, or sera samples are analyzed after the standard curve and after every ten patient samples. This same pattern of calibration and quality control placement is used in ICP-MS analyses.

17

tory quality control programs from the Centre de Toxicologie du Quebec (Urines certified for arsenic and cadmium); Centers for Disease Control and Prevention, College of American Pathology, and States of Pennsylvania and New York (whole blood).

2.3. Reagents 2.6. Patient samples

Stock solutions of As, Cd, Pb, and T1 were purchased from J.T. Baker Chemical Co. (Phillipsburg, NJ) as Baker Instra-Analyzed Atomic Spectral Standards. Stock solutions to prepare internal standards of Ga, Ge, Y, Rh, Pd, Re, Pt, and Bi were also purchased from J.T. Baker. Concentrated nitric acid was purchased from J.T. Baker as Instra-Analyzed Reagent Grade. Hydrochloric acid was purchased from Mallinkrodt, Inc. (Paris, KY) as AR Select grade. None of the reagents were purified further. Reagent quality water was produced by processing laboratory distilled water through a Barnstead NANOpure treatment system (Barnstead-Thermolyne; Dubuque, IA). Reagent grade water is routinely assayed for impurities by ICP-MS, ZGFAAS, and ICP-AES. 2.4. Laboratory ware

The following disposable laboratory items were used: Oxford pipette tips, no. 885-091341 (Monoject Scientific, Division of Sherwood Medical, St. Louis, MO); polystyrene 75 × 12 mm 5 ml tubes (no. 55.476) and 6.0ml screwcap vials (no. 61.542) (Sarstedt, Inc., Princeton, NJ). All items were used with no additional cleaning, 2.5. Certified controls

Specimens with pre-analyzed heavy metal concentrations were purchased from the National Institute of Standards and Technology, Gaithersburg, MD (NIST-SRM 2670 Toxic Metals in FreezeDried Urine, low and elevated concentrations); Bio-Rad, ECS Division, Anaheim, CA (Lyphochek Urine Metals Control, Level 2; Whole Blood Control, Level 2); Instrumentation Laboratory/Fisher Scientific Company, Orangeburg, NJ (Urichem Urine Chemistry Control, Human Level II). Survey samples obtained by participation in interlabora-

Patient samples sequestered for method comparison were selected at random from an average of more than thirty urine specimens and twelve whole blood specimens analyzed per day by Zeeman GFAAS. Urine samples were previously acidified with nitric acid to pH<2. Total volume excreted was measured and recorded for each 24 h urine collection. All samples were analyzed by ICP-MS and Zeeman GFAAS on the same day. 2.7. Standardization and analysis

In the final analytical format, a set of aqueous calibrating standards containing 1% HNO3 and concentrations of As, Pb, and T1 from 0 to 500 Ixg 1-I with Cd at 0 to 50 Ixg 1-1 were used to determine instrument response. These standards together with reagent blanks, urine controls, and patient samples were diluted 1:10 with a solution containing Ga, Rh, and Bi at 100 Ixg 1-~ in 1% HNO3. Each solution was vortexed and then placed in the autosampler rack. Blood samples were diluted as above but, immediately centrifuged for 5 min at 3200 rev rain-~. Supernatant solution was poured into another tube then placed in the autosampler rack. The cellular debris was discarded. Solutions were aspirated beginning with the blank, followed by the calibrating standards, controls, and then patient samples. Quality control samples were analyzed after every ten patient samples. Each solution was allowed to equilibrate in the nebulizerspray chamber for 75 s before analytical readings were taken. Peak Hop mode was used with a dwell time of 150 ms. Each reading is from 25 sweeps of the mass range. Isobaric correction for 4°Ar35C1 was made by measurement of the counts per second at mass 51 (16035C1). Because the intensity of 16035C1is about ten times the itensity of 4°Ar35C1 and is linear with

18

D.E. Nixon, T.P. Moyer/Spectrochimica Acta Part B 51 (1996) 13-25

increasing chloride concentrations, accurate corrections for the 4°Ar35C1 intensity at mass 75 were made. The corrected arsenic signal was obtained by subtraction of the calculated 4°Ar35C1 signal from the total signal measured at mass 75. With the exception of corrections for 1145n overlap with 114Cd, lJ2Sn with 112Cd, and 2°4Hg on 2°4pb, no other isobaric corrections or physical sample manipulations were performed. Signal suppression correction was performed by measuring Ga for As correction, Rh for Cd, and Bi for Pb and T1.

3. Results and Discussion

3.1. Isobaric Interferences Of the methods available for the correction of 4°Ar35C1 interference on 75As by concomitant chloride, the most desirable method is also the most transparent to the analyst. Routine methods that require extensive reagent manipulation, such as chromatographic separations or complex digestions followed by hydride generation, are prone to contamination and reagent addition errors. In addition, they also add time and imprecision to the method and exclude the determination of Cd, Pb, and T1. A computer augmented instrumental correction, if the degree of accurate correction can be attained, represents the ideal situation. Our first attempt at an instrumental correction used urine based calibration standards and yttrium as the internal standard combined with the classical 4°Ar37C1/82Se/83Kr correction for chloride interference. Results for eleven urines obtained as part of the Interlaboratory Program of the Centre de Toxocologie du Quebec gave arsenic results 20% higher than the certified concentrations. For the same specimens Zeeman GFAAS results were about 4% high. In addition to these certified urines, 190 urines routinely analyzed by Zeeman GFAAS were also analyzed by ICP-MS using the analytical conditions described above. The correlation data (y = 1.28x - 6.3; r = 0.9927; with Zeeman GFAAS as the independent variable) seems to suggest that ICP-MS results were also biased high for these real samples. We concluded that a better instru-

mental correction was needed even for a screening analysis. Because the classical 4°Ar37C1/82Se/83Kr correction does not yield adequate results, our next approach was to thoroughly investigate the 16035C1 correction empirically derived by Kershisnik et al. [20]. In their paper, chloride concentration was actually measured on patient urines and a relationship between the chloride concentration and intensities of 16035C1 and 4°Ar35Cl developed. Though this correction produced arsenic results closer to the target concentrations for Quebec urines, their results were still about 12% high. In our evaluation of this correction scheme we designed an experiment to also monitor other chloride containing masses and record the intensity changes with the addition of HC1 to patient urines. Our experiment, designed to determine if an exact relationship between added chloride and 4°Ar35C1 intensities could be defined in terms of any chloride containing polyatomic specie such as 16035C1 or 16037C1 was as follows: fifteen patient urines with volumes ranging from 320 to 2958ml were diluted 1:10 with nitric acid, containing yttrium as an internal standard at 100 ixg 1-l, so that the final nitric acid concentration was 1%. HC1 was added so that the final chloride concentrations ranged from 0 to 30 mM. Patient urines conceivably could contain chloride from this concentration range up to 100mM when diluted 1:10. We monitored the following polyatomic ions: 35C135C1, 35C137C1, 37Cl37C1, 16035C1, 16037C1, 4°Ar35C1, and 4°Ar37C1. In our trials only 16035C1, 16037C1, 4°Ar37C1 and 4°Ar35C1 gave linear responses with added chloride. All 15 reponse curves for each of the four polyatomic species listed above were parallel with each other. As might be expected the average slopes for each of the four polyatomic species were different from each other and approximately in the same ratio as the chloride abundance ratio. Six representative plots of urine response curves for 16035C1 and 4°Ar35C1 intensity with added chloride are shown in Figs. 1 and 2 respectively. We now focus on 16035Cl because, as might be predicted from the chloride abundances, the intensities of 16035C1 for all of the urines are about three times higher than 16037C1.

In both the classical 4°Ar37C1/82Se/83Kr correc-

D.E, Nixon, T.P. Moyer/Spectrochimica Acta Part B 51 (1996) 13-25

ICP-MS Response ot kioss 51 with Added Chloride to Urine

14 13

12 11 10 9 8

7 5 5 4

3 I

2

i 4-

I

I 8

I

i

1121116

i

2101214

2181

Chk~'ide Added to Urine (rnM)

Fig. 1. Response at mass 51 (16035C1) for added chloride to six urine samples.

ICP-MS Response ot Mo=s 75 with Added Chloride to Urine

1.3 12 1.1 1

0.9 0.8 0,7

0.6 0.5 04

o.~

o

I

i

4

i

I

8

i

~*~

I

~'6

I

2Lo

I

2',.

I

2'8

i

Added Chloride (raM)

Fig. 2. Response at mass 75 (75As + 4°mr35Cl) for added chloride to six urine samples.

tion and the empirical t6035C1 correction the polyatomic isotope response is used to mathematically generate a correction factor for the 4°Ar35C1 intensity contribution to the signal at mass 75. Because both corrections are based on intensity ratios, (16035C1 to 4°Ar35Cl and 4°Ar37C1 to 4°Ar35C1, respectively) it might seem logical to look at the intensities of both 16035C1 and 4°Ar37C1 ratioed to 4°Ar35C1 with added chloride for our 15 urines. The easiest way to accomplish this is to ratio the slopes of each polyatomic species to the response for

19

4°Ar35C1. The resulting data are presented in Fig. 3. It can be seen in Fig. 3 that for any given 4°Ar35C1 intensity the 16035C1 response is about 13 times greater than the 4°Ar37C1 response. The degree of variability of the 4°Ar37CL/4°Ar35C1 slope ratio is also somewhat greater than the 16035C1/4°Ar35C1 ratio. While the coefficients of variation are not radically different, the magnitude of variation in the correction factor is. Small fluctuations in 16035C1 intensity do not produce large variations in the mass 75 correction. The contribution of 4°Ar35C1 to the intensity measured at mass 75 can easily be eliminated by subtracting 0.07898 × lmass 5J from the intensity at mass 75. This is not the case for the classical correction where the factor is 1.04297. To test this refined 16035C1 correction nine Quebec urines were analyzed by first calibrating the ICP-MS with aqueous 1% nitric acid based standards containing 100 (l~g Ga) 1-1 as the internal standard. Samples were diluted with the acidinternal standard diluent. Arsenic concentrations averaged 7% higher than the target values. The Zeeman GFAAS results were again about 4% higher than the target values. The validity of this correction was also tested on 130 patient urines prepared as described above and analyzed after calibration with aqueous standards. The regression equation was y = 1.13x -6.0. It is evident that some bias still remains, but it has been reduced from 28% to about 13%. It should be noted that the ICPMS tends to yield higher arsenic results at moderICP-MS Slope I~otios for Chlorlae Additions ~2

09 -

\

08 07

ArCl(75)/ArCl(77)

Slope

-

1.04297

('V =

06

18 2 %

05 04 0.3

Slope ARC1(75)/0C1(51)

02 0.1 0

~ _._

=

II

i

__ 31

=

=

i

5~

.

~ t

= 0.07898 CV =

71

I

91

=

=

t

~J1

I

I I 3

%

ll3

I

1IS

Urines

Fig. 3. Slope ratios for the addition of chloride to fifteen urines.

20

D.E. Nixon, T.P. Moyer/Spectrochimica Acta Part B 51 (1996) 13-25

ate to high arsenic concentrations (300500 I~g/24 h) where the intensity from 4°AF35Cl should actually contribute little to the total intensity at mass 75. It is purely speculative to suggest that a fraction of the arsenic may be vaporized from the graphite furnace at this high concentration, but it is not unreasonable. From the foregoing experimental data we conclude that the 16035C1 correction gives more accurate results for arsenic than the classic 4°Ar37C1182Se/83Kr correction. The only caveat for the use of this or any other isobaric correction is that the element detected at the mass identical to that of the polyatomic molecule must not be present in concentrations large enough to interfere with the correction. For the 40Ar37C1182Se/83Kr correction, selenium must be measured accurately in order to make an accurate correction. For urine and serum references, approximate concentrations are less than 35 txg 1-I for urine and 95-165 txgl ~ for sera [29]. These normal selenium concentrations represent significant ion counts that must be measured accurately so that the selenium contribution to the 4°Ar37C1 signal can be accurately calculated. For the measurement of 16035C1 at mass 51, any residual vanadium must be taken into account. Normal vanadium concentrations are 0.87-8.55 txg 1-j in urine [30] and 0.017-0.139 I~g 1-1 in sera [31]. For urine, normal vanadium concentration is about eight times less than selenium; for serum, the factor is about 1400. We conclude that the vanadium contribution to the 16035Cl signal at mass 51 is not significant in normal patients. This conclusion was reached by Kershisnik et. al. [20] and is supported by this work. 3.2. Internal standardization

The primary purpose of internal standardization in ICP-MS is to correct for matrix included signal suppression or enhancement. The use of internal standards to correct for changes in transport from the bulk diluted sample to the detector has been reviewed [19] and guidelines for internal standard selection have been outlined [19,24--26]. Because matrix effects are dependent on the absolute amount of matrix present and not the ratio of matrix to analyte [ 19] and because the urine matrix

can vary significantly from one patient to another, three internal standard elements were chosen for the different mass ranges involved in our heavy metal screen. For our initial study described above yttrium, rhenium, and rhodium were chosen as internal standards based on the physiological rarity, mass, and ionization potential of each element compared to the analyte. Yttrium was chosen as an internal standard for arsenic (mass difference 14; ionization potential difference 3.4 eV); rhodium for cadmium correction (mass difference 10; ionization potential difference 1.5 eV); and rhenium for lead and thallium (mass difference ~21 and 18, respectively; ionization potential differences 0.5 eV and 1.8 eV, respectively). Results for urine analysis for arsenic, described in the previous section, were about 13% high even with the 16035C1correction and with yttrium as the internal standard. The correlation for cadmium versus Zeeman GFAAS was 1.074 - 0.03; r = 0.9830. From these data it would appear that rhodium, even with a mass difference of 10 u adequately corrects for the effects of the urine matrix on the cadmium signal. For lead, the correlation equation was 0.80x+ 10; r--0.9900. Note that ICP-MS results were about 20% low compared to Zeeman GFAAS. It appears that even though the ionization potential difference is only 0.5 eV, the mass difference between rhenium and lead of approximately 21 u does not adequately correct for matrix suppression. In this first study no positive thallium urines were encountered. Because urine results for arsenic were still high and results for lead were low compared to Zeeman GFAAS, the logical next experiment seemed to be an evaluation of selected internal standard elements for all four analytes. For our evaluation Bio-Rad Lyphocheck Urine Metals Control, Lot No. 44502, Lyphocheck Whole Blood Control, Lot No. 57602, NIST-SRM 2670, and Seronorm Whole Blood III, Lot No. 205053 were analyzed by diluting the samples 1:10 with a diluent containing 1% nitric acid and Ga, Y, Re, Bi, Rh, Ag, In, and Sb each at 100 Ixg 1-I concentration as potential internal standards. Aqueous calibrators with concentrations ranging from 0 to 500 ~g 1-1 were prepared for As, Pb and TI. For cadmium the concentration range was 0-50 Ixg 1-1 In this experiment

D.E. Nixon, T.P. Moyer/Spectrochimica Acta Part B 51 (1996) 13-25

Ga and Y were used as internal standards for As; Re and Bi for Pb and T1; and Rh, Ag, In, and Sb for Cd. For whole blood samples the cellular residue after acidification was removed by centrifugation. Concentrations for the four analytes determined by applying the listed internal standard correction are shown in Table 2 for certified urine controls and in Table 3 for the certified whole blood controls. The most dramatic effect of internal standard choice appears to be with lead and thallium. Concentrations for both lead and thallium are nearly identical to the target values when bismuth is the internal standard. Precision is also better. This improvement in accuracy is directly attributable to a reduction in the mass difference between the analytes (Pb and T1) and bismuth (mass = 209) versus rhenium (mass -- 187 + 185). In the case of arsenic and cadmium essentially no improvement in accuracy was attained with Ga for arsenic or Ag, In, and Sb for cadmium. When Ga was used as the internal standard, precision of arsenic determi-

21

nation improved. For cadmium no accuracy or precision improvement was observed. In fact, for silver as the internal standard, the precision was much worse than Rh. Rhodium appears to be the best choice because the others appear to be poorer choices. Silver does not seem appropriate because of the large excess of chloride in urine. For indium the mass difference is very small, but the ionization potential is far less than cadmium. The final internal standard ensemble then is Ga for As; Rh for Cd; and Bi for Pb and T1.

3.3. Quality control The secondary use of internal standardization in ICP-MS is to correct for long term instrumental stability or drift. While the raw counts for each element measured in a standard solution appear to remain relatively constant from week to week, some drift with time can be expected if for no other reason than changes in the detector response, nebu-

Table 2 Comparisons of analyte concentrations and coefficients of variation measured on certified urines for different internal standards Bio-Rad Level II Conc./(ixg 1-~) Arsenic Target value None Ga Y

147 192 140 140

NIST-SRM 2670 CV/%"

Conc./(~g 1-~)

CV/%"

9.9 1.0 1.1 3.5

480 626 491 491

20.8 0.6 0.7 2.1

Cadmium Target value Rh Ag In Sb

13.1 11.7 12.5 I 1.5 9.9

9.9 1.1 4.5 0.9 0.8

88 80 85 78 67

3.4 0.8 3.3 0.8 0.7

Lead Target value Re Bi

55.6 44.5 55.9

10.0 1.7 1.0

109 86 110

3.7 1.7 0.7

Thallium Target value Re Bi Coefficient of variation.

207 164 206

9.9 I. 1 1.0

Not certified -

D.E. Nixon, T.P. Moyer/Spectrochimica Acta Part B 51 (1996) 13-25

22

Table 3 Comparison of analyte concentrations and coefficients of variation measured on certified whole bloods for different internal standards Bio-Rad Lyphocheck

Seronorm whole blood Conc./(ixg 1 ~) Cadmium Target value Rh Ag In Sb

a

Conc./(ixg 1 ~)

CV/%"

not certified

12.4 11.4 12.0 11.1 9.6

Lead Target value Re Bi

CV/%"

3.7 15.3 3.0 2.8

663 533 611

0.2 1.0

240 192 233

10.4 0.3 0.7

Coefficient of variation.

Table 4 ICP-MS seven day quality control study Conc./(txg 1-') Target ± SD

ICP-MS ± SD

Arsenic Uri-chem Bio-rad NIST 2670 1 NIST 2670 II Seronorm

258 --- 38 147 ± 14 (60) " 480 ± 100 25 added

313 ± 6 140 ± 4 81 ± 1 491 _+ 3 31.3 ± 0.7

Cadmium Bio-rad NIST 2670 I NIST 2670 II Seronorm

13.1 ± 1.3 (0.40) a 88 + 3 12.4

Lead Uri-chem Bio-rad NIST 2670 I NIST 2670 II Seronorm Lyphocheck

193 ± 21 55.6 -+ 5.6 (10) a 109 ± 4 671 ± 18 240 ± 25

250 ± 5 56.0 _~_-1.0 6.8 ± 0.1 110 ± 0.7 611 ± 4 236 ± 3

Thallium Bio-rad

207 ± 20

207 _+ 5

a Information only.

11.7 ± 0.3 0.52 ± 0.04 80 ± 0.6 12.7 ± 0.2

lization process and sampling of the plasma. In order to ascertain how effective our choices of internal standards are at correcting for matrix effects and instrumental drift, seven quality control samples with certified element concentrations were analyzed each day for seven days. All aqueous calibrators and samples were diluted 1:10 with the internal standard mixture in 1% nitric acid. t6035C1 was used for isobaric arsenic correction. Mean and standard deviation data together with target values are shown in Table 4. In general, the ICP-MS standard deviations are better than those of the certificate values. Accuracy in all cases was acceptable, but arsenic results were higher than certified values for all but the Bio-Rad urine. It is obvious that some error in isobaric correction still remains.

3.4. Survey samples Whole blood and urine specimens are routinely issued by regulatory agencies to check the proficiency of analysis of subscribing laboratories. The purpose of the survey samples is to detect errors in analysis technique and methodology so that accurate results are reported for patient samples. It is imperative that any new technique meets the requirement of accurate results for survey specimens. Failure to correct deficiencies in

D.E. Nixon, T.P. Moyer/Spectrochimica Acta Part B 51 (1996) 13-25

23

Table 5 Correlation of ICP-MS

and Zeeman

GFAAS

results for whole blood and urine survey samples with target concentrations

Urine

Whole blood

ICP-MS

regression

equations Arsenic

y = 1.10x - 4.7

r = 0.9833

No surveys

r = 0.9891

y = 0 . 8 7 x + 0.5

n = 26 Cadmium

y = 0.98x - 0.5

Lead

n = 22

n = 28

No surveys

y = 0 . 9 9 x + 1.1

r = 0.9826 r = 0.9980

n = 36 ZGFAAS

regression

equations Arsenic

y = 0.81x + 24

r = 0.9314

No surveys

Cadmium

y = 1.04x - 0.8

r = 0.9887

y = 0 . 9 1 x + 0.1

r = 0.9788

Lead

No surveys

y = 1.15x - 5 3

r = 0.9961

Table 6 Regression equations for the comparison

of ICP-MS

with Zeeman

Urine regression equations

Arsenic

y = 1.13x - 6.0

GFAAS

for whole blood and urine samples Blood regression equations

r = 0.9684

No data

r = 0.9839

y = 1 . 0 1 x - 0.1

n = 130 Cadmium

y = 0.94x - 0.0 n = 130

Lead

y = 0.96x -

1.6

r = 0.9879

n = 130 Thallium

y = 1.09x -

r = 0.9962

n = 110 y = 0.98x + 0.4

r = 0.9954

n = 98 11.0

r = 1.0000

No data

n=10

analysis can mean denial of licensure to analyze particular samples such as whole blood for lead. Because graphite furnace atomic absorption spectrophotometry is the technique most used for the determination of As, Cd, and Pb in urine and blood, it is useful to compare results generated by both Zeeman GFAAS and ICP-MS with the target values for survey samples. In our comparison, calibration curves were generated for ICP-MS using aqueous acidic standards with internal standard elements added as described above; all dilutions were 1:10. For Zeeman GFAAS analysis of arsenic and cadmium in urine the calibration curve was generated by recording absorbances from standards prepared as aqueous acidic standards added to a

urine matrix. Whole blood lead and cadmium calibration curves were generated from aqueous standards without matrix addition. Table5 shows regression equations for results by each technique compared to the target values. All ICP-MS and Zeeman GFAAS results were within the acceptable limits of the target values. Note that for urine arsenic the ICP-MS slope is about 10% high and the ZGFAAS correlation has a 20% low slope with a large intercept. With correlation coefficients of 0.9314 for ZGFAAS and 0.9833 for ICP-MS there appears to be more scatter to the ZGFAAS results than the ICP-MS results. On average ICP-MS gave results 6.8% higher than target for arsenic urine; the ZGFAAS results were 4.5% below tar-

24

D.E. Nixon, T.P. Moyer/Spectrochimica Acta Part B 51 (1996) 13-25

get. F o r whole b l o o d lead the Z G F A A S intercept is negative but the slope is 15% high. F o r Z G F A A S the average results were 2.1% high c o m pared to target concentrations. The I C P - M S results were only 0.5% b e l o w target. 3.5. Patient samples

Before the introduction o f I C P - M S as the instrument used for routine determination o f As, Cd, Pb, and T1, patient samples were a n a l y z e d by both techniques to access the correlation o f I C P - M S with our standard Z e e m a n G F A A S technique k n o w n to produce accurate results for real samples. Regression equations for As, Cd, and Pb c o m p a r a tive analysis on 130 urines originally analyzed as metal screens are shown in Table 6. Regression equations for individual urines acquired for and positive for thallium analysis are also listed in this table. W h o l e b l o o d c a d m i u m and lead samples are also listed. A b s e n t are equations for whole b l o o d arsenic and thallium. Half-lives o f these elements in whole b l o o d are very short. Significant concentrations o f these elements in whole b l o o d w o u l d indicate acute intoxication by these elements within hours of b l o o d sampling; it is rare to see positive b l o o d samples for arsenic or thallium except in such cases. It can be seen that correlations between I C P - M S and Z e e m a n G F A A S were excellent.

4. Conclusions F r o m our investigation o f I C P - M S as a technique for screening urine and whole b l o o d for As, Cd, T1, and Pb we conclude the following: 16035C1 can be used more effectively to i m p r o v e the accuracy o f the isobaric correction o f 4°Ar35C1 on the arsenic signal at mass 75 than the classic isotope correction using 4°Ar37C1/82Se/83Kr. W e also conclude that aqueous acidic standards and simple sample dilution with Ga, Rh, and Bi as internal standards p r o d u c e accurate results. In addition I C P - M S can perform the same analyses as Z e e m a n G F A A S in o n e - h a l f the time with o n e - h a l f the technical effort.

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D.E. Nixon, T.P. Moyer/Spectrochimica Acta Part B 51 (1996) 13-25 [16] H. Vanhoe, C. Vandercasteele, J. Versieck and R. Dams, Determination of iron, cobalt, copper, zinc, rubidium, molybdenum, and cesium in human serum by inductively coupled plasma mass spectrometry, Anal. Chem., 61 (1989) 1851-1857. [17] H.T. Delves and M.J. Campbell, Measurements of total lead concentraions and of lead isotope ratios in whole blood by use of inductively coupled plasma source mass spectrometry, J. Anal. At. Spectrom., 3 (1988) 343-348. [18] M.A. Vaughan, A.D. Baines and D.M. Templeton, Multielement analysis of biological samples by inductively coupled plasma-mass spectrometry. II. Rapid survey methods for profiling trace elements in body fluids, Clin. Chem., 37 (1991) 210-215. [19] E.H. Evans and J.J. Giglio, Interferences in inductively coupled plasma mass spectrometry. A review. J. Anal. At. Spectrom., 8 (1993) 1-18. [20] M.M. Kershisnik, R. Kalamegham, K.O. Ash, D.E. Nixon and E.R. Ashwood, Using 16035C1 to correct for chloride interference improves accuracy of urine arsenic determinations by inductively coupled plasma mass spectrometry, Clin. Chem., 38 (1992) 2197-2202. [21] W.B. Barnett, V.A. Fassel and R.N. Kniseley, An experimental study of internal standardization in analytical emission spectroscopy, Spectrochim. Acta Part B, 25 (1970) 139-161. [22] G.J. Schmidt and W. Slavin, Inductively coupled plasma emission spectrometry with internal standardization and subtraction of plasma background fluctuations, Anal. Chem., 54 (1982) 2491-2495. [23] F.J. Feldman, Internal standardization in atomic emission and absorption spectrometry, Anal. Chem., 42 (1970) 719-724.

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[24] J.S. Crain, R.S. Houk and F.G. Smith, Matrix interferences in inductively coupled plasma-mass spectrometry: some effects of skimmer orifice diameter and ion lens voltages, Spectrochim. Acta Part B, 43 (1988) 1355-1364. [25] J.J. Thompson and R.S. Houk, A study of internal standardization in inductively coupled plasma-mass spectrometry, Appl. Spectrosc., 41 (1987) 801-806. [26] F. Vanhaecke, H. Vanhoe, R. Dams and C. Vandercasteele, The use of internal standards in ICP-MS, Talanta, 39 (1992) 737-742. [27] E.R. Denoyer, D.E. Nixon and D. Hilligoss, ICP-MS Heavy metal screening in human urine for clinical diagnostics: scope, limitations and comparison of conventional and flow injection sample introduction, FACSS XX, Detroit, Michigan, 17-22 October 1993. [28] D.E. Nixon, G.V. Mussmann, S.E. Eckdahl and T.P. Moyer, Total arsenic in urine: palladium-persulfate versus nickel as a matrix modifier for graphite furnace atomic absorption spectrophotometry, Clin. Chem., 37 (1991) 1575-1579. [29] D.E. Nixon and T.P. Moyer, Is palladium a better matrix modifier than nickel for the Zeeman graphite furnace determination of selenium in serum and urine? FACSS XX, Detroit, Michigan, 17-22 October 1993. [30] P. Bermejo-Barrera, and Bermejo-Martinez, J.A. Cocho de Juan, Determination of vanadium in urine by electrothermal atomisation atomic absorption spectrometry, J. Anal. At. Spectrom., 2 (1987) 163-166. [31] J. Versieck and R. Cornelis, Normal levels of trace elements in human blood plasma or serum, Anal. Chim. Acta, 116 (1980) 217-254.