dry and wet ashing techniques in analyses for zn,cu,mn and fe in hair 1986

8/2/2019 Dry and Wet Ashing Techniques in Analyses for Zn,CU,Mn and Fe in Hair 1986

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CLIN.CHEM. 32/5, 739-742 (1986)

CLIN ICA L CHEM IS TRY , V ol. 3 2, N o. 5, 1986 739

Dry - and We t-Ashing Techn iques Compared in Analy ses fo r Z in c, Copper,M an ganese, and Iro n in H airJames K. Friel and C hau D . N gyuenPreparation o f h air specimens for trace-m etal analyses isrout inelydone by we t- o r d ry -a sh ing. We t- ash ing is mo retim e consum ing than dry-ashing and can be dangerous. W ewished to determine if dry -ash ingwas a su i tab le a lte rna tiveto wet -ash ingwi th HCIO4:HNO3or HNO3 alone in prepar ingha ir for measurement o f z inc , copper, i ron ,and manganeseby atomic absorption spectroscopy. Concentrations of Zn ,Cu, an d M n w ere n ot d iffe ren tly a ffected in h air th at was dry-or w et-ashed. A nalytical recovery of th ese elem en ts ad ded tohair sam ples ranged from 102 to 108% ; day-to-day C Vs w ere18 MWcm

was obtained from t he B ar ns te ad p ur if ic at io n system (Barn-D e pa rtmen t o f Biochemistry, Memor ia l U n iv er si ty o f Newfound-l and , S t . J ohn s, N ew foundl and , Canada, A 1B 3X 9.Rece iv e d Oc to b er 2, 1985; a cc ep te d J an u ary 16 , 1986.

stead Co., Boston, MA 02132). W e washed the hair sam pleswith a 10 mLIL solution of a non-ionic detergent (Aca-tionox; American Scientific Products, JL 60085) in de-io niz ed w ate r. Fo r dry-ashing we used Suprapur-gradeH NO 3 (E . M erck , D ain stad t, F .R .G . ); f or wet-ashing w e u sedreagent-grade H NO 3 an d H C1O 4 ( Fis he r S cie ntific C o., F airL aw n, N J 07410).To avo id con tamina t ion , we soaked all glassware in dilute(50 m LIL ) HNO3 for two days, and then rinsed with de-io niz ed w ate r. F or a ll injections in to th e g ra ph ite fu rn ac e weused trace-elem ent-free disposable polypropylene pipettetip s (F ish er S cie ntific Co., no. 22 34 190-1).

Standards. Zn, Cu, Fe, and Mn standards were preparedby diluting 1 g /L stoc k solutions ( Fi sh er S cie nti fi c Co. ) withde-ionized w ater. W orking standards at two concent ra ti on sfor both Zn an d Cu were 1 an d 4 tg/m L, for Fe 0.5 and 5pg/mL, and for Mn 5 and 15 ng/mL.Procedures

S pe ctr op ho to me te r s ettin g. For f lam e p ro ce du re s, w e usedth e glass impact bead in the assays of Zn, Cu, an d Fe. TheZ n h ollow -cath od e lam p current w as 15 m A; wavelength,213.9 ma; slit w idth , 0.7 nm . The Cu hollow -cathode lampcm-rent was 15 mA; wavelength , 324.8 nm ; slit w idth , 0.7nm . The Fe hollow-cathode lamp (multi-elem ent lamp)current was 30 mA; w avelength, 248.3 nm slit width, 0. 2nm . For all f la me a na ly se s th e in tegration time was 2 a;results were calculated from th e average of three read in gs.Flameless analyses for M n were perform ed w ith deuteriuma rc b ack grou nd c orrec tion ; th e M n hollow-cathode lam pcurrent was 15 mA, wavelength , 279.5 nm ; slit width, 0. 2m a.

G ra ph ite fu rn ac e p ro gr am . The basic settings used fo r t hegraphite furnace were: Dry: 1 30 # {1 76 }C ,amp 20 a, hold 60 a;Char 1 07 0 # {1 76 }C ,ange 30s, hold 30s; A tom ize : 24 00 #{ 176 }C ,am p0 a, hold 5 s; C lean: 27 00 # {1 76} C,am p 1 s, hold 2 a. The argong as f lo w was 300 m L/m in du ring th e entire procedure exceptfor 4 s during atom ization , when it was 0 m lim in .

Collection of h air sam ples. For the control study, w ecollected hair from various areas of the head of one of us(J . K . F .), cut it with s ta in le ss -s te el s cis so rs , an d stored it inpolyethylene tubes. The entire fibers (not segm ents) w erein clu ded in th e sam ples.

Using s ta nd ard iz ed p ro ce du re s (12), we a ls o c ol le ct ed hairsamples from 20 women who ha d recently given birth toinfants with neural tube defects or to norm al, healthy full-term infants. F or b ot h groups of w om en, h air was cut aboveth e nape of the neck, as close to th e scalp as possible. Onlythe 1-2 cm of hair closest to the scalp was retained foranalysis.

Preparation o f h air samples. W e placed the hair samplef or t he control study in a 500-mL a cid -w as he d E rle nm ey erflask an d added 20 0 mL of th e A catio nox so lu tion . T he flaskw as covered with Parauilm an d agitated at room tempera-tare for 60 mmwith a mechanical sh ak er. W e th en filtered


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Mean (SD), L9/g dry wt

Percent r ec ov er y, m e an (SD)

Mean (SD), g /g dry w tZn Cu Mn

Certified value 852 (14) 63(3.5) 17.5 (1 .2) 195 (34)TreatmentDry 85 9 (1) 65 (0 .4) 18 (0 .8) 202 (7)HNO3 864 (2) 72 (3) 20 (1.6) 192 (4)HNO3/HCIO4 8 35 (1 00 ) 58 (8) 15.9 (0 .8) 218 (9)740 CLINICA LCHEMISTR Y,Vol .32,No.5, 1986

th e hair through an a cid -w ash ed B uc hn er f un ne l c on ne cte dto a vacuum aspirator and rinsed the sam ple several tim esw ith a to tal volume of 2 L of de-ionized water beforecovering it an d leaving it to dry overnight at 55 #{176}Cn adrying oven. F ou rte en p or tio ns of this con trol sa mp le o f h airwere transferred t o a ci d-washed 15-rnL porcelain crucibles,and 28 portions were placed i n a ci d -wa shed 250-m L P yrexbeakers. A ll sam ples weighed between 100 and 200 mg.

D ry -ashi ng . Ten of th e control hair samples were ashed ina m uffle furnace (Fisher Isotem p m uffle furnace, M odel184) for 12 h at 4 50 # {1 76 }C ,f te r o ve n- dr yin g at 250 #{176}Cor 2 h.After th ey h ad cooled for 1 h, w e removed the sam ples fromt he f ur na ce an d added five drops of Suprapur HNO3. Onehour later, w e returned th e samples to th e m uffle fu rn ace,where they were heated at 250 #{176}Cor 2 h, then ashed at4 50 # {1 76 }Cor an additional 8 h (11). W e again let the samplescool for 1 h, then added 1 mL of S uprapur 11N 03, in 4 mL ofwater, to each beaker. A fter 30 mm , we decanted th esamples into 25-m L volumetric flasks, washed th e b ea ke rswith de-ionized water, and used th e c omb in ed washings tobring th e sam ples to volum e.

Wet -ashing. W e placed 10 c on tr ol s am p le s of hair in 250-mL wide-mouthed Pyrex beakers, and added 10 mL ofreagent-grade H NO 3 to each beak er an d let these sit over-n ight. Ten other control samples of hair were placed insim ilar beakers and left to digest in 5 mL of HNO3 over-n ight. The follow ing morning we added 10 mL ofHC1O4:HNO3 (1:4 by vol) (4) to the latter 10 beakers. A llsamples were covered w ith watch glasses an d refluxed at120-140#{176}Cor about 4 h until the digests were clear and 2-3 m L of acid rem ained. A fter cooling for 1 h we decanted th edigested samples into 25-m L volum etric flasks an d broughtto volum e w ith added rinsings of d e- io niz ed w ate r.

Assessing accuracy and precision. Because no certifiedhair stan dard is currently availab le, w e used U .S . N at io na lBureau of Standards c er tifie d o ys te r t is su e (S ta nd ard R efe r-ence M aterial no. 1566) containing know n am ounts of Z n,Cu, Fe, and M n to assess th e a cc ur ac y of the methods. Fora na ly tic al -r ec ov er y e xp er im e nts , we added a k no wn a mo un to f e ac h metal to fou r portions of c on tr ol h air for each of thedry-ashing, HN O3-ashing, and H C1O4:H NO 3-ashing proce-dures. W e also added kn ow n am ounts o f e ac h metal to puresolutions, which were analyzed w ithout an y interveningashing step s.

M easuring m etals in h air s am ple s. For assay of Zn, Cu,an d Fe, w e aspirated wet- and d ry -d ige sted sam ples dire ctlyinto th e flame. For Mn det erm in a ti on s , we in je ct ed samplesmanually in to th e g ra ph ite tube w ith an E pp en dor f pipette.Sam ple size was 20 i.L , and each sample was analyzed intriplicate. W e repeated th e assays of 10 samples on differentdays to assess b etw ee n-r un v ar ia tio n. W e a ls o r ep ea te d th eassay of on e sample 10 t imes during th e same day to assessw ith in -ru n v ar ia tio n.

S ta tis ti ca l a na ly si s. D ifferences betw een hair concentra-tions in each group w ere assessed b y o ne -w ay analysis ofvariance (13).ResultsM easured concentrations of zinc, copper, and m anganese

(Table 1) in h air treated by dry- and wet-ashings did notdiffer. T he ir on c on ce nt ra tio n measured in hair that w asdry-ashed was lower than that in h air that was wet-ashed.Recovery of know n am ounts of metals added to puresolutions was: 99 1% for Zn, 103 3% for Cu, 96 10%

Table 1 . Concen tr atio n s o f Z In c, C o pp er, M a ng an es e,a nd Iro n In C on tro l H air Treated by Dry- a nd We t-AshingsZn Cu Mn

Dry 216 (4) 44 (1) 0.74 (0.06) 27 (9 )HNO3 216 (28) 42 (5) 0.82 (0.11) 38 (6)bHNO3:HCIO4 227 (31) 44 (6) 0 .77 (0.12) 41 (l2)LValues with differentsuperscrip ts are s ign i fi can t ly d i ffe ren t f rom eachother at p < 0.0 5. n = 10 .f or M n , an d 10 4 1% fo r F e. Recovery of known am ounts ofadded m etals to hair control sam ples w ere com parable fo rz in c, c o pp e r, an d iron in h air treated b y d ry - a n d we t- as hi ng .W e found high concentrations of manganese in h air tre ate dw it h HC 1O4:HNO3, com pared w ith the amount of manga-nese added (T able 2).Zinc, copper, iro n, and m anga nese con cen tratio ns o f o ys-te r tis su e tr ea te d by dry ashing w ere w ith in th e range ofNBS-certifled values (Table 3). The CVs for 10 co ntrol h air

s am ple s r e- an aly ze d on five different days were


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Zn Cu Mn17 7 (26) 32 (1) 2.07 (3.5)19 3 (30) 30 (21) 0 .88 (0 .85)

CLINICA LCHEMISTR Y,Vol .32, No.5, 1986 74 1

in determ ining if dry ashing of hair samples provides asatisfactory alternative to wet-ashing with eitherHC1O4:HNO3 or HNO3 alone.

Zinc. Concentrations of Zn in hair treated by threed if fe re nt a sh in g p ro ce du re s d id not differ (Table 1 ) , sugges t-ing that dry ashing is a suitable preparation for analysis forZn i n hair . V o la til iz at io n losses d ur in g a sh in g h ave n ot beenreported at the ashing temperature we used, but some Znm ay be retained o n c ru ci ble walls (20). W e di d not find anylosses of Zn after dry-ashing, because w e e va po ra te d al lsamples with HN O3, a method known to im pr ov e r ec ov er y(4). Recovery of added Zn (102 7% , Table 2) a nd a cc ur ac yof oyster tissue analysis (Table 3) supports th e su itability ofth e dry-ashing treatm ent prior to analysis of Zn in hair.

Copper. Concentrations of Cu in c on trol ha ir sam ples didnot differ am ong the three treatm ents (Table 1). Recovery(108 13%) a nd a cc ur ac y of Cu determinations after dry-ashing were com parable to those results obtained by wet-d igestion (Tables 2 an d 3) . Contamination with Cu fromporcelain crucibles has been reported previously (11) an dm ay explain the slightly higher analytical recovery f or d ry -ashed samples than was fo un d for w et-a sh ed s am ple s. Cu isnot volatile at the ashing temperature w e u sed (20); howev-er , there have b een re ports (4) of variable and increasedlosses of Cu as the temperature of d ry -ash in g in cre asesabove 4 00 # {1 76 }C .ry-ashing of hair sam ples is a reliablet reatment in preparation for C u analysis.

Manganese. Concentrations of M n in hair treated b y d ry -ashing did not differ significantly from t ho se r es ult s ob -tain ed after w et-a sh in g (T ab le 1 ). M n v olatilizatio n duringdry ashing is unlikely below 550 -6 00 #{ 17 6}C ,lthough som eretention of M n on crucible walls has b ee n re po rte d (21).Recovery of M n is improved with HNO3 (22), and therecovery from porcelain crucibles during dry-ashing in thisexp erim ent w as ac ceptable (108 5% , T ab le 2). Recovery ofMn after wet-digestion by H NO 3:H C1O 4 was high (120 10% , T able 2). This may be attributed to pipetting error orcontam ination, which is a risk because of the very lowcon cen tration s of M n in hair as compared w ith th ose of othermetals (Table 4). W e do not th ink that the slightly low ervalues for M n obtained during dry-ashing are ascribable toeither volatilization or retention of m anganese on crucib lewalls. O ther workers (4) have used ashing aid s for m an ga -nese analyses in tissues, but th is extra step would not beuseful, considering the low concentrations of manganese inhair a nd th e c or re sp on din gly increased p robability of con -tam ination . Recently , G uillard et al. (23) rep orted on m an-ganese analysis in hair after w et-d ig estion in T eflo n b om bs.T h e r ep or te d within-run CV (4% vs 8% ) an d r ec ov er y (1 04 9.2% vs 108 5% ) of M n w ere lower than ours. The slightlylower findings (23) may not justiir th e expense of the m in i-autoclave (current cost of th e P erkin -E lm er n o. 078486,$140.00 U .S.) in com parison with th e cost of porcelain orsilica cru cib les ($6 .00 U .S ., F ish er Scien tific C o.).

T ab le 4 . C on ce ntra tio ns o f Z in c, C op pe r, a ndManganese In the HaIrofMothersofInfantsithNeural Tube Defect or Normal Healthy ChIldren

Neur al t ub e d ef ec t ( n = 10 )No rmal (n = 10 )

Mean (SD) , pQ/g dr y w t

Iron. Measured concentrations of Fe in hair were signiiI-cantly low er in s am ple s tre ate d by dry-ashing as com paredwith w et-ashing (Table 2). Fe concentrations of oyster tissueanalyzed after preparation by the dry-ashing technique(Table 3) were w ith in the e xpe cted ran ge (202 7 tg/g vs19534 jtg/g, c ertified value). M atrix d ifferenc es m ay haveaccounted fo r lo we r measured Fe concentration in hair ascompared with the measured Fe concentration in oystertissue. Some Fe may volatilize during dry-ashing, butp re vi ou s r es ea rc he rs have reported no Fe losses at 50 0 or600#{176}C24). R etention losses for Fe have also b ee n r ep or te dd ur in g d ry -a sh in g (27), although our analytical recovery byth e d ry -a sh in g te ch niq ue (110 9% ) w as com parab le to thato f th e HNO3 digestion m eth od (114 14% ) and high er th anth e r ec ov er y by the HC1O4:HNO3 digestion technique (100 11 % , T a ble 2). Recovery of Fe a ft er d ry -a sh in g w as h ig he rthan that found for Zn, Cu, or M n. P re vio us r es ea rc he rsh ave also re porte d higher recovery for Fe as compared withthat found for Zn an d Cu in liver (19) an d hair (5). Dry-ashing m ay not be suitable prior to determining Fe in hair.Dry-ashing for F e h as p reviou sly b ee n r ec omm en de d onlyfor large and (or) fa tty s am ple s (20). W e found that treat-ment with HNO3 gave results comparable w ith those ob-tained by H C1O 4:H NO 3 d igestion (T ab le 1). Treatm ent w ithHNO3 has previously been reported to be suitable for Feanalysis in h air (25). It should be noted t ha t, i f t emper at ur esexceed 1 50 # {1 76 }Cu rin g w et-a sh in g, som e Fe may be lost (4).

W atting and Wordale (4 ) reported that concentrations ofm etals in fish tissu e a fter w et. or d ry-ashin g were compara-ble . They suggested that contam ination during w et ox id a-tio n m ay resu lt from th e ad dition of reagents to samples, th econtam ination frequently exceeding th e amounts in thes am ple its elf. W e used Suprapur-grade HN O3 on ly d urin gd ry -a sh in g, b ec au se each s am p le r eq uir ed only 1 m L. Supra-pur H C1O 4 is not available an d the large amount of acidrequired for wet digestion (10 m L per s am p le ) p re clu de s th eu se of S up rapu r HNO 3 b ecau se of th e e xp en se . Thereforeblank values obtained w ith reagen t-grad e acid during w et-ashing were higher than Suprapur blank values obtainedduring dry-ashing. B lank values from dry ashing of 18b la nk s, c al cu la te d in m icrogram s per gram of dry hair, w erelow for Zn (0.04), Cu (0 .04), and M n (0 .02), but were high forFe (0 .85 ) -about 5-8% o f th e usual F e con cen tration in hair.A fu rther d ifficulty d urin g w et oxidation is t he p re pa ra ti onof acid-matched sta nda rd s, b ecau se the am ount of acid thatis e vap orated differs fo r e ac h sample.W e suggest that dry-ashing is a suitable treatment fo rhair before analyses for Zn, Cu, and M n. Wet-digestion w ithHNO3 rather than H C1O 4:H NO 3 is acce pta ble before th eanalysis of Fe in hair. Z n, C u, and M n concentrations in hairanalyzed after dry-ashing are presented in Table 4 formothers who gave birth to infants w ith neural tu be defectand mothers of normal healthy children . The results suggesta d if fe re nc e in trace-m etal status of th ese two groups. Alarger stu dy of th ese tw o g ro up s is curre ntly in p ro gress.

W e thank D r. C lark Fraser a nd H il lo ry V a va so ur fo r collectingth e h air sam ples, Drs. Gene H erzberg and L A . W . Feltham fo rtheir helpful com ments, and C arol M urph y for p re pa rin g th ismanu sc ri pt . F in an ci al s uppor t w as receiv ed fro m th e PresidentsNSERC Fund.

References1. Anderson J. W et d ig es tio n v ers us d ry a sh in g f or t he analysis off ish t is sue f or t ra ce m et als . A t Absorpt Newsi 1972;1l :88-9.


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2. Giron HC . Comparison betw een dry ashing a nd w et d ig estio n inth e preparation o f p la nt ma te ri al f or a tomic absorp ti on analyses. AtAbsorpt Newsl 1973;12:28-9.3. Isaac RA , Johnson WC. Co ll ab or ativ e s tu dy of w et and dryashing techniques fo r th e elem en tal analysis o f p la nt tissue bya tom ic a bs or pt io n s pe ctr op ho tom et ry . J Assoc O ff Anal Chem1975;58:436-40.4. Wattin g H R, W ord ale IM . Comparison o fw e t a nd d ry a sh in g fo rth e analysis of b io lo gi ca l m a te ri al s by A AS. In: T he analysis ofbiological materials, B utler L RP , ed., Oxford: P er gamo n P re ss ,1979;69-80.5. Harrison WW, Y u ra eh er J P, Benson CA . The determination oftrace elem ents in human hair by a tom ic a b so rp ti on spectroscopy.C lin C him A cts 1969;23:83-91.6 . H ilderbran d D C, W hite DH . Trace-element analyses in hair anevaluation. C lin Ch em 1974;20:148-51.7. S tevens BJ, Determination o f a lum ini um , copper an d zinc inhum an hair. Atom Spectr 1983;4:176-8.8 . P e te ri ng HG , Y eager D N, W itherup S O. T race m etal content ofh air I. Zinc an d copper c on te nt o f hum an hair in relation to age an ds ex . A rc h E nv ir on H ea lth 1 97 12 3:2 02 -7 .9. Eads EA , Lambdin CE . A survey o f t ra c e metals in h um an h air.E nv ir on R es 1973;6:247-52.10 . S chro ed er H A, N aso n A P. T race m eta ls in hu ma n h air. J InvestDermatol 1969;53:71-8.11 . S alm ela S , V uo ri E , K ilpioJO . T he e ffect of wa sh in g p ro ce du re son trace element content of human hair. Anal Chim Acts1981;125:131-7.12 . Friel JK , G ib so n RS , Balassa R, W atts JL . A comparison o f t hezinc, copper a nd mangan es e s ta tu s o f v er y l ow b ir thwe igh t pre-terman d full-term in fan ts d uring th e first tw elv e m on th s. A cts PediatrScand 1984 ;7 3 :5 96-601 .13 . Nie NH , Hull CH, Jenkins JG , S teinbierner K , B ent DH .

S ta ti st ic al p ac ka ge f or t he s oc ia l sciences. N ew Y or k: McG raw H illInc. , 1 9 75 .14 . G ib so n R S. The tra ce m eta l s ta tu s o f s om e C anadian full terman d l ow birthwe ight infants at on e year o f a g e. J R ad io an al C h em1982;70:175-89.15 . L ake r M . O n d eterm ining trac e e lement l ev el s in man : th e u se sof b lo od a nd h air . Lancet 1982;ii:260-2.16. Kopito L , Biley AM , Shw achm an H . C hronic plum bism inchildren. J Am Me d A ss oc 1969,209:243-8.17 . Val ko vi c V . Analyses of biological material f or t ra c e e lement su s ing x -r a y spectroscopy. B oca R aton, FL : CRC Press, 1980.18 . Cookson JA , P ill in g FD . T ra ce e lem en t d is tr ib ut io n across th ediameter of hum an hair. Phys Med B io l 1 97 5; 20 :1 01 5- 20 .19 . C ubeils M JS , D e la Guardia C irugeda M , Q uerada LA . R a piddetermination o f copper , iron an d zinc i n l iv er biopsies. At Spectroec1984;5:217-22.20 Tsalev DL . Atomic absorp ti on spectrometry in occupational an de nv ir onm en ta l h ea lt h practice. Vo lum e IL D e te rm in atio n o f i nd i-vidual ele me nts . B oc a Raton, F L: C RC P ress, 1983.21 . G orsuch V F. The destruction of organic matte r. O xfo rd : Perga-m on P ress, 1970.22 . Casey CE. Concentrations of som e trace elem en ts in humanan d cows m ilk Proc Univ Otago M ed Sch 1976;54:7-8.23 . Guillard 0, Brugier JC , P iriou A , M enard M , Gombert J, ReissD . Improv ed d et erm in at io n of m anganese in hair by u se of a m in i-autoclave a nd f la in ele ss a tom ic a bs or pt io n spectrometry w ith Z ee -man background correction; an evaluation o f unexpo eed subjects.Clin Chem 1985;30:1642-5.24 . K oirtyohann S R H opkins CA . L oss of trace metals duringashing of b io lo g ic al ma te ri al s. An al ys t 1976;101:870-5.25 . T anak a T , H ayashi Y , Funakaw a K , Ishizaw a M . S im ultaneousdetermination o f F e and M n in hum an hair by graphite-furnace tw oc ha nn el AAS . N ip po n K ag aku K aishi 1 98 1;1 :1 69 -1 74 . I n Japanese,with abstract i n E n gl is h.

dry and wet ashing techniques in analyses for zn,cu,mn and fe in hair 1986

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