Discussion
Many factors influence measurement of Ca2, e.g., sampling conditions, sample type, and sample handling. If
plasma or whole blood is to be analyzed, effects on the Ca2’
caused by the anticoagulant (which is usually heparin) must
be considered. For adults, Ca2 is preferentially determined
in venous serum; thus an anticoagulant is obviated. However, in the neonate, capillary blood is generally preferable,
and thus heparin is needed to prevent clotting after sampling. When we used commercially available pre-heparinized capillary tubes, clotting still occurred. Therefore, we
routinely add 0.9 int. unit of extra heparin to the tubes
before sampling (1).
To clarify the effect of this additional heparin on Ca2’, we
compared values for Ca2* obtained with the above-described
sample handling with those after adding the same volume of
sodium chloride instead of the extra heparin. The added
sodium chloride produced the same lowering effect on Ca2
as did heparin. Evidently the effect of our heparin addition
was entirely ascribable to dilution. Consequently, this effect
was EVF-dependent and more pronounced at high EVF
values. It could be noted, however, that Ca2 in blood is a
part of a calcium equilibrium system in which more than
half of the calcium is complex-bound. Therefore, the dilution
effect was not as large as expected if only the volumes were
taken into account. For example, addition of 10 pL to a 1501iL sample volume, where 53% is plasma, should result in a
dilution effect of Ca2 from 1.24 to 1.09 mmol/L, but in our
study this addition resulted in a measured Ca2’ of 1.19
mniol/L (Table 1).
The systematic error caused by dilution can be compensated for (see Figure 1). At normal values for EVF and Ca2,
however, this error is hardly of clinical importance, but for
infants with very high EVF (>70%) and (or) Ca2 near the
lower normal reference limit it is more notable and should
be considered.
Ca2 and EVF were not correlated in our
study, and the changes in Ca2 during the first five postna-
tal days were larger than any systematic error caused by the
additive or by changes in EVF. Hence we propose that the
increase in Ca2 during this period (8) is of physiological
origin and not an artifact of the sampling method.
We conclude that heparin added to capillary
samples
causes an EVF-dependent systematic error in Ca2 by
dilution. This should be considered when EVF values are
high and (or) Ca2 is near the lower reference limit. We
recommend a compensatory adding of 0.09 minolfL to the
measured actual Ca2 value at EVF >70%. The increase in
Ca2 seen in normal infants during the first postnatal week
evidently is of physiological origin and not an effect of a
decreasing hematocrit in combination with heparin additive.
References
1. Larseon L, FinnatrOm 0, Nilsaon B, Ohman S. Evaluation of
Radiometer ICA 1 as a routine instrument for serum ionized
calciumand its applicationfor wholebloodcapillary samplesfrom
newborn
infants.
Scand J Clin Lab Invest 1983;43:21-6.
2. LadensonJH, Bowers GN. Free calcium in serum. I. Determination with the ion-specific
electrode, and factors affecting the results.
Clin Chem 1973;19:565-74.
3. Urban P, Buchman B, Scheidegger D. Facilitated determination
of ionized calcium. Clin Chem 1985;31:264-6.
4. Biswas CK, Ramos JM, Kerr DNS. Heparin effect on ionized
calcium concentration. Clin Chim Acta 1981;116:343-7.
5. Wezzoli G, Elli A, Palazzi P, et al. Limits for use of heparin in
ionized calcium determination. Ric Clin Lab 1984;14:535-8.
6. Robertson WG, Maschall RW. Ionized calcium in body fluids
[Review]. Crit Rev Clin Lab Sci 1981;15:85-126.
7. Matoth Y, Zaizov R, Varsano I. Postnatal changes in some red
cell parameters. Acts Paediatr Scand 1971;60:317-23.
8. Nelson N, FinnstrOm 0, Larsson L Neonatal reference values
for ionizedcalcium, phosphate and magnesium. Selection of refer-
ence populationby optimality criteria. Scand J Clin Lab Invest
1987;47:111-7.
9. McGovern J, Jones A, Steinberg A. Hematocrit of capillary
blood.N Engl J Med 1955;253:308-12.
CLIN. CHEM. 35/3, 488-490 (1989)
Micro-QuantityTissue Digestionfor Metal Measurements by Use of a Microwave Acid-Digestion
Bomb
James R. P. Nicholson,1M. Geraldine Savory,1 John Savory,1’2and Michael R. Will&3
We describe a simple and convenient method for processing
small amounts of tissue samples for trace-metal measurements by atomic absorption spectrometry, by use of a
modified Parr microwave digestion bomb. Digestion proceeds rapidly (90 s) in a sealed Teflon-lined vessel that
eliminates contamination or loss from volatilization. Small
quantities of tissue (5-100 mg dry weight) are digested in
high-purity nitric acid, yielding concentrations of analyte that
Departments of’Pathology, 2Biochemistry, and3 Internal Medicine, University of Virginia Health SciencesCenter, Charlottesville, VA 22908.
ReceivedSeptember16, 1988; acceptedDecember 27, 1988.
488 CLINICALCHEMISTRY, Vol. 35, No. 3, 1989
can be measured directly without further sample manipulation. We analyzed National Institute of Standards and Technology bovine liver Standard Reference Material to verify the
accuracy of the technique. We assessed the applicability of
the technique to analysis for aluminum in bone by comparison with a dry ashing procedure.
Addhional Keyphrases: samplepreparation
atomic absorption
spectrometry aluminum
bone
trace elements
.
Trace-metal measurements
in tissues by atomic spectroscopictechniques require preliminary digestion of the sample, most often by heating in strong acids. However, conventional open-vessel techniques require considerable time (4-
48 h) and can lead to loss of volatile analyte and environmental contamination.
Recently, microwave
radiation
has
been used to heat the acid. When it is used with sealed
microwave-transparent vessels, temperatures above the
boiling point of the acid are produced. This drastically
shortens the time needed for digestion (1-3) and facilitates
the preparation of large numbers of samples for analysis.
We have applied this technique to the digestion of small
amounts of tissue such as biopsy specimens by modif’ing a
commercially available microwave digestion bomb. The
modified Parr bomb offers distinct advantages over other
available microwave digestion systems. The 4.5-mL internal
volume of the modified Teflon liner and the correspondingly
low volume of nitric acid used (1-2 mL) provide reliable, fast
digestion while minimizing
dilution of the already low
metal concentrations frequently found in physiological samples. This digestion system provides a convenient and inexpensive means of performing difficult digestion of tissue
specimens and does not require special oven venting.
Materials and Methods
Apparatus and Reagents
The commercially available microwave digestion bomb is
equipped with a 23-mL Teflon liner (Parr Microwave Acid
Digestion Bomb, Model 4781; Parr Instruments Co., Moline,
IL 61265). The modified liner is made of tetrafluoroethylene
(Teflon) machined to the same outside dimensions as the
original 23-mL liner to accept the original 0-ring sealed lid,
but has an internal well 1.11 cm in diameter, with a conical
bottom. A Teflon screw, seen as a button protruding through
the top of the bomb shell, retains the compressible relief disc
and acts as a pressure indicator (Figure 1). The microwave
oven we used is a Kenmore Model 566-8868620 (Sears,
Roebuck and Co., Chicago, IL 60684) with power consumption of 1400 W, at 2450 MHz. It has a transparent observation window. A laminar-flow hood capable of producing
Class 10 air (10 particles per cubic meter) is required for all
specimen handling. To dry the tissue samples, we used a
box-type muffle furnace (Blue M Electric Co., Blue Island, IL
60406) and Teflon beakers covered with Teflon watch glasses. Acid-washed Pyrex beakers with Pyrex watch glasses
were used in the muffle furnace to ash bone samples.
Fig. 1. Cross sectionof Parr microwave add-digestion bomb with
modified(low volume)wellTeflonliner
Positionof original(Parr lnstr.Co.) well is shown (
) for comparison
Doubly distilled (sub-boiling method in quartz apparatus)
concentrated nitric acid was purchased from the National
Institute of Standards and Technology [NIST (formerly
National Bureau of Standards)], Gaithersburg, MD 20899,
and stored in a Teflon container. De-ionized water (18 mfl
cm’
resistance) was used for all dilutions.
Atomic absorption spectrometric analyses were performed
with a Model 5100 instrument utilizing an electrothermal
graphite atomizer with a stabilized temperature platform,
equipped with Zeeman background correction (PerkinElmer Corp., Norwalk, CT 06856). For zinc analysis, we
used the instrument’s flame atomizer with deuterium background correction. Spectrometric quantification of selenium
was by method of additions. All other elemental quantifications were determined by comparing spectrometric data
with the appropriate aqueous calibration curves.
Specimens
Bovine liver Standard Reference Material (SRM 1577a;
NIST) is available in powder form. Human cortical bone,
approximately
5 mm square (about the size of an iliac crest
biopsy sample), was obtained from various sites of an
excised femoral head. For the comparison
studies we digested four portions by the microwave technique and four others
by dry ashing. Other tissues were collected in acid-washed
plastic implements and containers, and were stored at
-20 #{176}C
until analysis.
Procedures
Drying. One-hundred-milligram
samples of SRM 1577a or
human cortical bone were dried to constant weight at 85#{176}C
for 48 h in weighed Teflon beakers covered with Teflon
watch glasses, in the muffle furnace. Specimens of liver,
kidney, muscle, and brain to be analyzed for metals can be
handlod the same way.
Microwave bomb digestion. Transfer -50 mg of the dried
sample from the Teflon beaker directly into the Teflon bomb
liner, and determine the exact transferred sample weight by
difference in weight of the Teflon beaker. To this sample,
add 1000 pL of de-ionized water and 1000 pL of concentrated nitric acid. Prepare blanks that contain only acid and
water. After capping the liners, seal them in the bomb shell.
Place the assembled units, one at a time, in the center of the
microwave oven and irradiate at full power for 45-90 s, the
time depending on sample type. Remove the bomb from the
microwave oven and let it cool at room temperature for at
least 20 miii. At this point, the outer shell can be opened if
no pressure is evident, and the Teflon liner can be removed.
Remove the liner cap carefully and recombine any condensate with the digest in the sample well. Dilute samples and
blanks after digestion as necessary for analysis to ensure
that sample concentrations fall within each element’s calibration range. Continuous visual monitoring of the pressure
indicator button is required throughout the irradiation
period, because excessive pressure can build up rapidly.
Discontinuing power to the oven to stop temperature increases immediately stops any further increase in pressure.
The compressible relief disc is designed to vent pressure
over 10.5 MPa, avoiding rupture of a completely sealed
container. Other workers using different forms of microwave
bombs have reported ruptured containers (4, 5). An indicator button rise of 1 mm (approximately 3.5 MPa increase
according
to the manufacturer’s operating instructions) is
sufficient pressure to stop microwave heating of the sample.
Even with this cautious approach to heating, samples are
CLINICAL CHEMISTRY, Vol. 35, No. 3, 1989 489
and the desirable qualities of a completely sealed
container are maintained.
Dry ashing. For comparison, we also processed four portions of dried and weighed cortical bone by dry ashing, as
follows: Place the samples in acid-washed 100-mL Pyrex
beakers, cover with Pyrex watch glasses, and dry-ash in the
muffle furnace at 550 #{176}C
for 48 h. After cooling, add 3.0 mL
of water and 1.0 mL of concentrated nitric acid to the
residual ash. Heat the contents of the loosely covered
beakers to dryness on a hot plate, dissolve the white residue
in 1.0 mL of dilute (2 mIJL) HNO3 (NIST grade), and
analyze by electrothermal atomic absorption spectrometry.
Take empty beakers through the procedure as blanks. After
digestion, dilute samples and blanks with water as necessary for analysis.
digested,
Table 1. Analysis of Bovine Liver (NIST SRM 1 577a)
Content,
Element
Zn
Fe
ig/g
dry wt.
Certified
123±8
194 ± 20
Cu
Co
Experimentalb
121±3
193 ± 12c
158±7
0.21 ± 0.05
Pb
Al
Se
0.135
153±2
0.19 ±
002d
0.015
0.141 ± 0.007
2#{176}
2.15 ± 0.04
0.71 ± 0.07
0.75 ± 0.07
a.fl..,
estimateduncertainties
are based on judgmentand representan
evaluationofthecombined effects ofmethodimprecision,
possible systematic
errorsamongmethods,and materialvariabilityforsamplesweighing250 mg
or more.”Certificateof AnalysisforSRM 1577a. Meanof duplicateanalyses
±
b
forseparatelydigested samples;n = 2 unlessnoted. cn = 3#{149}
d n = 4.
The
concentrationof aluminumin SAM 1577a is a recommendedvalue only.
Results and DIscussion
We used the acid-digestion microwave technique to analyze SRM bovine liver for Al, Pb, Co, Cu, Fe, Se, and Zn. The
experimental results were compared with certified NIST
values. Additionally, we compared bone Al values obtained
by the microwave method with those obtained by a standard
dry-ashing
technique. The technique has also been used in
our laboratory for the determination
of Al in brain, liver,
kidney, and muscle specimens.
Table 1 summarizes the results obtained for NIST-certifled bovine liver (SRM 1577a) analyzed by the microwave
acid-digestion procedure. The experimental results agree
well with the certified (recommended) values. The MIST
Certificate of Analysis states that a minimum of 250 mg of
material should be used for any analytical determination for
comparison with the certified value. In most instances, we
used <60mg of material. However, our results indicate that
homogeneity of this sample was not a problem.
The microwave digestion procedure for bone analysis was
evaluated by comparison with a well-established dry-ashing
technique. We chose to measure aluminum in bone because
of the increased
incidence of increased concentrations
of it in
osteodystrophy
in dialysis patients. Moreover, aluminum is
highly susceptible to contamination
from the environment
and thus presents a special challenge to the analyst. Because of the nonhomogeneity of aluminum in bone, we
analyzed four portions of cortical bone by each method in an
effort to obtain a representative value. Bone Al results
obtained by both methods are reported in Table 2.
Sample and blank contaminations for bone Al analysis
were minimized by using the sealed Teflon container. Also,
use of the microwave procedure decreased the aluminum
measured in the blank solution by 10-fold, the measured
absorbance values of the blank for the dry-ashing technique
being 26-54% of the absorbances for the prepared samples
vs a minimal 2-3% with the microwave technique. Finally,
there were no spurious outliers in the sample analyses after
microwave digestion, in contrast to those noted with the dryashing technique.
490
CLINICAL CHEMISTRY, Vol. 35, No.3, 1989
Table 2. AlumInum Content In Cortical Osteomalaclc
Bone Samples Compared after MIcrowave Digestion
and Dry-Ash Techniques
Al
concentration,,LgIgdry
Microwave digestion
30.5
42.2
42.8
weight
Dry-ash technique
40.0
33.4
32.3
29.3
Mean±SD(n=4)36.2±7.3
132.4#{176}
Mean ± SD(n = 3) 35.2 ±4.2
aOutiler;valuenotincludedin determinationof mean.
The microwave digestion system reported here evidently
provides an inexpensive, efficient way to prepare samples
for analysis by atomic absorption spectroscopy. In the past
year we have used this method to prepare various samples of
pathological interest, including brain, bone, and other tissuesfor aluminum content; eye lenses for calcium; and liver
needle-punch biopsies for iron analysis for hemochromatosis
assessment.
Supported in part by Grant ES04464 of the National Institute of
Environmental Health Sciences.
References
1. Kingston HM, Jassie LB. Microwave energy for acid decomposition at elevated temperatures and pressures using biological and
botanical samples. Anal Chem 1986;58:2534-41.
2. Aysola P, Anderson PW, Langford CH. Wet ashing biological
samplesin a microwave oven under pressure using poly(tetrafluoroethylene) vessels. Anal Chem 1987;59:1852-3.
3. Kingston HM, Jassie LB. Microwave acid sample decomposition
for elemental analysis. J Res Natl Bur Stand 1988;93:269-74.
4. Gilman L, Grooms W. Safety concerns associated with wet
ashing samples under pressure heated by microwave
energy
[Letter]. Anal Chem 1988;60:1624.-5.
5. Aysola P, Anderson PW, Langford CH. Response to safety
concerns[Letter]. Anal Chem 1988;60:1625.