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Determination of gallium in tumour-affected tissues by means of spectroscopic techniques
Analytica Elsevier
Chimica Scientific
Acta,
136 (1982)
Publishing
225-231
Company,
- Printed
Amstcrdnm
in I’he
Nethrrlsnds
I~I~TE:RhlINA’I’ION OF GALLIIJM IN TUMOUR-AI~‘FKCXED MEANS OF SPECTROSCOPIC TECHNIQUES A Comparative Study
S. CAROI.I*.
A. ALII\ION’I’I
I.ahoratorio di Tossicologia. Roma (Italy)
and P. DELIX
TISSUES
I3Y
FEMMINE
Istitu to Superiorc
di Sanitci.
Vialc
Neginu
E1l~nu -399. 00 16 I
S. K. SliUKLA I.uboratorio di Chimica Stu--ionc. Homa (Italy) (Received
18th
September
Nuclearc
tic1 C.N. It.. Cuselln
I’ostalc
10. OOIG
. dlontcrotoncio
1981)
21 comparative investigation was carried out on the 5uitirbility 0f atomic absorption spectrometry and of c-mission spectrography with arc and hollo\v-cilthodc cscitrrtion sources for determination of gallium (pg g”) in biological samples. The three methods give rcliablc msults. Hollow-cathode to ;I lesser extent by matris efiects
emission
spectrography
and to be more
precise
The potential usefulness of gallium-containing diagnosis and therapy is well documented [ l-61.
was found than
to be influenced
the othcar two
formulations
techniques.
for
tumour
Iiowever, it is generally acknowledged that none of these gallium preparations can be consiclcred to be universally applicable. It would seem that a fundamental roie is played by the type of anionic species present, the nature of which contributes to making the formulations more tumour-specific and rapidly efficacious. The mechanism of gallium uptake is still unknown. To elucidate this, it is essential, as a preliminary step, to accurately determine the distribution of gallium in the various organs after parentcral administration. From this point of view the most suitable procedures use spcctrochemical methods because of their reliability and versatility. In order to evaluate the applicability of spectroscopic techniques for gallium in biological samples, three tcchniqucs (each based on different physical phenomena) werct selected. These were atomic absorption spectrometry, and atomic emission spcctr(lgriiphy with two different excitation sources, an arc and a hollow cathode. ESPERIhlEN’I’AL
Preparation of the samples Morris hepatoma 3924 A cells were inoculated in the right hind legs of AC1 rats (6 groups of animals, each of 5 subjects); likewise, mammary tumour 0003-2670/82/0000~000Q~s0~.
75 CJ 1982
Elwvicr
Scientific
Publishing
Company
cells were injected into mice (10 groups of 5). Seven days after inoculation, all groups but one (to be used as a control for both species) were treated every other day for 3 weeks with gallium nitrate solutions containing various concentrations of sodium citrate. The amount of gallium thus administered totalled in all cases 8 mg kg* of body weight, whereas that of sodium citrate ranged from 0 to 30.5 mg kg”’ of body weight. After spontaneous death of the animals, tumour-affected tissues as well as healthy organs (lung, heart, liver, spleen and kidney) were escised. The weight ranges of the various organs are shown in Table 1. All samples were kept frozen until treatment could be started. The organic samples were wet-ashed in concentrated nitric acid (65%, Merck Suprapur). Depending on the weight of the sample, lo-50 cm’ of acid was used. The mixtures were evaporated over a small flame until a clear solution was obtained on cooling. No filtration was found to be necessary_ The procedure resulted in complete destruction of the organic material and conversion of gallium to its nitrate_ The volume of the solutions thus obtained was 2-3 nitric acid and subcm3. These were made up to 10 cm3 with concentrated sequently diluted (1 + ‘9) with double-distilled water. Three identical aliquots of each were finally taken for determining gallium_ There is no serious risk of external contamination by gallium. Nevertheless, thorough rinsing of all g!assware with high-purity 1 M nitric acid was effected prior to use, and manipulations of the samples were minimized. Analyses were carried out by means of the three spectroscopic techniques mentioned after preliminary experiments for optimizing the working conditions for each. 411 measurements were carried out in triplicate and averaged. TGS
Atomic
absorption
spectrometry
(a.a.s.)
Measurements were made with a Perkin-Elmer model 430 atomic absorption spectrometer and a carbon furnace, connected to a model 56 recorder. The furnace tubes were of pyrolytic graphite to allow a larger number of injections (ca. 100) with each tube. High-purity argon was used to purge the furnace at 900 ml min-’ in an interrupted flow mode. Although preliminary results showed that background correction was
TABLE
1
Range of organ weights
Organ
Lung Heart Liver
in rats. and mice
Weight (g) --_---_._-
~-
RatS
Mice
1.3-2.1 1.1-1.6 8.5-l 1.0
0.2-0.4 0.1-0.1s 1.1-1.5
Organ
Spleen Kidney Tumour
Weight (g) Rats
Mice
0.8-1.6 1.9-2.4 2.1-8.5
0.24.4 0.1*.25 0.4-1.4
superfluous during the atomization stage, it was decided to apply it merely to minimize recorder scale deflection caused by smoke formation during the thermal decomposition phase. Eppendorf pipettes were used to inject 10 ~1
of
solution
furnace. The main operating parameters nm; lamp current, 15 mA; furnace conditions, lOO”C, 5 s, rate 2; 8OO”C, 5 s, rate 2; 25OO”C, 7 s, rate 0 (gas interrupt). Calibration graphs were constructed from the peak heights obtained from solutions containing 10, 50, 200, 1000 and 1500 ng Ga ml-’ in 10% nitric acid. Differences in salt content of the various organs generally, and sometimes remarkably (as in the case of liver .samplcs), affected the reliability of measurements and thus led to erratic results. This could be ascertained by plotting calibration graphs for gallium in solutions obtained from different organs and comparing them with the graphs pertaining to solutions containing only nitric acid and gallium nitrate. In all the cases investigated. the slope of the calibration plots was significantly different to that obtained with gallium nitrate and nitric acid. Therefore it was concluded that for each type of sample it is necessary to resort to the standard addition method, which proved successful for the elimination of some systematic errors. An effective standard reference material, however, would afford more consistent data. adopted
were:
manually
into
wavelength,
the
287.4
Arc emission spectrography :\ PGS-2 plane-grating spectrograph
(VEB Carl Zeiss Jena, 650 grooves mm-i, blazed at 5” 35’. Kbert mounting, 20-pm entrance slit, intermediate diaphragm 3.2) was used with the arc emission source, which was equipped with an arc generator (Optica, Milan). The procedure adopted was based on the standard addition method. because matris effects were noticeable, though to a lesser extent than with a.a.s. A lo-cm’ aliquot of sample solution was first added with an equal volume of a solution containing 10% sodium sulphate and 1% sulphuric acid as well as various aliquots of the standard gallium solutions. The mixtures thus obtained were dried under ix. irradiation and calcined at 350°C for 3 h. The residues were accurately homogenized and introduced in the crater of graphite electrodes (Ringsdorff RW 0 52 anode cup), and arced at 8 A for 35 s, to ensure complete consumption of the added sample. The gallium line at 294.4 nm was preferred because it was practically free from interference from other elements and reflected concentration variations reliably. Spectra were photographed on Kodak Spectrum Analysis No. 1 plates. Plates were developed in a Kodak solution D-19 for 4 min at 20 ? O.l”C. The blackening values were measured by a microdensitometer MD-100 (Jenoptik Jena, GDR), capable of evaluating blackening as great as 3.2 with an error of less than 2%. Correction for background required the preliminary elaboration of a sensitometric struction of standard addition
curve, which was also calibration graphs.
necessary
for the con-
228
Hollow-cathode
emission
spectrography
The instrumentation employed was a vacuum spectrograph SPV lm/800 equipped with a l-m radius concave grating with 1200 grooves mm-*, blazed at 5” 52’, in a Paschen-Runge mounting, with a 30+m entrance slit, a HVG 2 electric source and a hollow-cathode lamp, all supplied by WV-Prtiisionsmessgetitc GmblI (8031 IIechendorf/Pilsensee, \V. Germany). High-purity argon at 550 Pa was used as the filler gas, because both its ionization potential and atomic mass are sufficiently high to assure an appreciable sputtering of cathodic material. The lamp current, was 400 mA, and the voltage 275290 V. A 4-min exposure of SA No. 1 emulsion was used for photographing
the spectra, supported on film. Films were processed under the same con-
ditions previously mentioned and the blackening at. the 417.2-nm gallium line was read with the MD-100 microdensitometer. The most suitable material for preparing holIow cathodes was stainless steel, from the point of view of both excitation stability and analysis sensitivity. Materials such as graphite and aluminium were also tested, but for various reasons were considered inconvenient and therefore not used further_ Preliminary tests were carried out to ascertain whether matrix effects were relevant or not.. Results showed that calibration graphs obtained by the standard addition method in the presence of different matrices gave no appreciable variation in slope and intercept. so that one calibration graph would suffice for all analyses. The nitric acid solutions obtained from the mineralization of the organic samples were introduced as 400+1 aliquots by means of a micropipette into the hollow cathode held vertically with its open end up. The cathodes were dried under i-r. irradiation and calcined at 500°C for 30 min. The 417.2-nm gallium line was chosen as the others were either not sufficiently intense or were masked by lines emitted by the cathodic material. Visual inspection after the discharge of the inner part of the electrode revealed that uniform sputtering occurred over the walls of the back half of the cathode bore, including the surface at the closed end. Under the experimental conditions of current intensity and gas pressure adopted for the discharge, it is in fact the lower part of the inner surface that is expected to contribute more effectively to the sputtering process. Therefore, a regular and efficacious progress of the discharge can be assumed for the present determination. RESULTS AND DISCUSSION
Typical gallium concentrations determined in the organic samples are set. out in Table 2. These values are representative of those groups of rodents (one for rats and one for mice) which showed the longest survival time. In the case of rats this corresponded to gallium formulations containing the largest amount of sodium citrate (equal to 30.5 mg kg-l body weight), whereas for mice the most suitable concentration of sodium citrate was found to be 7.5 mg kg-* _ The meaning of these data, however, will not be
229 TABLE
2
Average concentration Organ
of gallium found in the various organs (~g g- ‘)
Atomic absorption spectrometry ---I Rat
Lung Heart Liver Spleen Kidney Tumour
0.36 0.17 3.5 1.6 1.8 1.5 ~
Arc emission spectrography -Rat .----_-._-
Mouse _____ 0.4 1 0.09 2.09 0.78 1.48 0.96 __--- -
-
0.38 0.18 4 .o 1.7 2.0 1.7
.-Mouse
--Hollow-cathode emission spectrography .-Rat -
0.44 0.10 2.2 0.90 1.70 1.03 _.-
0.36 0.18 3.6 1.6 2.0 1.5 ..-
-
-.-
hlousc ---. 0.39 0.07 2.2 0.85 I.45 1.03
examined in this paper, because the main purpose here is to elucidate the applicability of spectroscopic methods to the determination of gallium in biological material. In order to make a reliable comparison of the analytical capabilities characterizing the three techniques, all determinations were repeated many times (never less than 10 measurements for each sample), and the results averaged. The parameters which allow an overall evaluation of each method are given in ‘Fabic 3. As far as sensitivity is concerned, in a.a.s. this is usually the concentration corresponding to 1% absorption and not simply the slope of the linear plot of the analytical signal as a function of concentration. Nevertheless, in this study it ‘cvas decided, for the purpose of comparing results, to calculate the sensitivity of the a.a.s. determinations on the latter basis, as done for the
TABLE
3
Characteristics samples hfcthod --Atomic absorption Arc emission Hollow-cathode emission
of spectroscopic
techniques
__ Uctcction limit“ (ppm)
__-_...__ Precisionb (6)
0.005
-I
0.1 0.01
9 2
for the determination
of gallium in biological _-
-..--
_.-._.
Retative sensitivityE
hlatrix t?ffect
98
0.55
High
10.1 102
0.20 1 .OO
Intermediate Low
hlean
recovery (S) _--___---
---
of variation, as calcutatcd on the basis of 10 measurements “3~ background. “Coefficient for a.a.s. and 12 for the other two m&hods. cSopc of calibration graph divided by standard deviation.
230
other two spectroscopic methods. The procedure described by Grant [7] was adopted in order to normalize sensitivities espressed with different units of the analytical signal. A comparison of the sensitivities pertaining to different analytical methods is in fact not directly possible because the signaf is
espressed by units typicaf for the technique considered (absorbance for a.a.s., blackening units for spectrography). Any analytical procedure can, however,
be essentially characterized by precision and sensitivity, both quantities being related to the particular units adopted. Therefore, the quantity obtained by dividing sensitivity by the standard deviation for a given method gives results independent of any particular system of units and, as a consequence, allows a meaningful comparison of data referring to different techniques. The accuracy of the methods was tested by doping, with 0.2 and 1 mg 1-l. the sample solutions obtained after wet ashing of biological material from rodents not treated with gallium and therefore free from detectable levels of this element. The corresponding mean recoveries are reported in Table 3.
Two principal considerations can be made on the basis of the results obtamed in this study. All the spectroscopic methods tested afforded reliable measurements and proved to be suitable for determining gallium in biological samples. Hollow-cathode emission spectrography appears to be superior to the other techniques insofar as it is relatively free from matrix interferences. Sputtering sources such as this are also characterized by high reproducibility and favourabIe detection limits [S, 91, although the latter are not as good as in a.a.s. Generally speaking, emission spectrographic techniques require a greater number of manipulations, this being a not negligible drawback and usually prolonging the time necessary for the analysis. In the case of hollowcathode emission spectrography, however, the possibility of utilizing a single calibration plot for all determinations (as a consequence of the freedom
from matrix effects) largely counterbaiances sample manipulation.
the disadvantages of longer
Comparison of the different features of the three techniques indicates that hollow-cathode emission spectrography is the most suitable, even though a.a.s. presents some remarkable advantages insofar as rapidity of analysis and detection limits are concerned. From this point of view it must be acknowledged that spectrographic methods cannot compete with a.a.s. because such tedious and time-consuming steps are involved as processing of emulsions and evaluation of spectrograms. This aspect, however, does not really affect
the intrinsic advantages of hollow-cathode emission spectrography which depend essentially on the discharge properties and not on how the signal is detected. It is conceivable, therefore, that direct-reading spectrometers would greatly improve the speed of determination for the hollow-cathode emission source without altering the basic features of this source. This fact
should be borne in mind while considering the analysis times which are, for a series of 12 determination, of the order of 2, 4 and 4 h for a.a.s., arc emission spectrography and hollow-cathode emission spectrography, respectively,
23 1
(12 determinations of esposures which
were chosen because they correspond can be taken on the same film or plate).
This paper was September 1981.
presented
in part
at
the
XXII
to
CSI/Sth
the
number
IC?\S.
Tokyo,
REFERENCES 1 C. I,. Edwards and R. L. iinycs. J. Nucl. 2 It. Wang, U. K. ‘Ilerner, D. English, A. Nucl.
Biol.
Xlcd.,
7 (1930)
Med.. 10 (1365) 103. A. Noujaim, B. C. Lcntlc
3 hl. M. Hart and Il. H. Adamson. Proc. Natl. Acad. Sci. U.S.A.. -I hl. hl. Iiart. C. F. Smith, S. T. Ynncy nnd K. Ii. Adnmson, 5 G 7 8 9
and
J. R. Itill.
In:. J.
9.
68 (19Tl) .J. Natl.
1623. Cancer
Inst.. 47 (1971) 1121. R. L. Hayes, J. Nucl. Med., 18 (1977) 7.10: 20 (1979) 938. S. K. Shukln. L. Castelli, I. Blotta. P. Delle Frmminc. S. Caroli and A. Alimonti, 3rd Int. Symp. Radiophnrm. Chem., St. Louis, USA, 1 G-20 June 1980. C. I,. Grant, Atomic Absorption Spectroscopy, ASTIM SW 443. 1969. p. 37. S. Caroli. A. Nimonti and I’. Dcllc Fernmine, Spcctrosc. I.ctt.. 12 (1979) 87 1. h. Alimonti, S. Caroli and N. Violnntc. Spcctrosc. Lctt., 13 (1980) 313.
Chimica Scientific
Acta,
136 (1982)
Publishing
225-231
Company,
- Printed
Amstcrdnm
in I’he
Nethrrlsnds
I~I~TE:RhlINA’I’ION OF GALLIIJM IN TUMOUR-AI~‘FKCXED MEANS OF SPECTROSCOPIC TECHNIQUES A Comparative Study
S. CAROI.I*.
A. ALII\ION’I’I
I.ahoratorio di Tossicologia. Roma (Italy)
and P. DELIX
TISSUES
I3Y
FEMMINE
Istitu to Superiorc
di Sanitci.
Vialc
Neginu
E1l~nu -399. 00 16 I
S. K. SliUKLA I.uboratorio di Chimica Stu--ionc. Homa (Italy) (Received
18th
September
Nuclearc
tic1 C.N. It.. Cuselln
I’ostalc
10. OOIG
. dlontcrotoncio
1981)
21 comparative investigation was carried out on the 5uitirbility 0f atomic absorption spectrometry and of c-mission spectrography with arc and hollo\v-cilthodc cscitrrtion sources for determination of gallium (pg g”) in biological samples. The three methods give rcliablc msults. Hollow-cathode to ;I lesser extent by matris efiects
emission
spectrography
and to be more
precise
The potential usefulness of gallium-containing diagnosis and therapy is well documented [ l-61.
was found than
to be influenced
the othcar two
formulations
techniques.
for
tumour
Iiowever, it is generally acknowledged that none of these gallium preparations can be consiclcred to be universally applicable. It would seem that a fundamental roie is played by the type of anionic species present, the nature of which contributes to making the formulations more tumour-specific and rapidly efficacious. The mechanism of gallium uptake is still unknown. To elucidate this, it is essential, as a preliminary step, to accurately determine the distribution of gallium in the various organs after parentcral administration. From this point of view the most suitable procedures use spcctrochemical methods because of their reliability and versatility. In order to evaluate the applicability of spectroscopic techniques for gallium in biological samples, three tcchniqucs (each based on different physical phenomena) werct selected. These were atomic absorption spectrometry, and atomic emission spcctr(lgriiphy with two different excitation sources, an arc and a hollow cathode. ESPERIhlEN’I’AL
Preparation of the samples Morris hepatoma 3924 A cells were inoculated in the right hind legs of AC1 rats (6 groups of animals, each of 5 subjects); likewise, mammary tumour 0003-2670/82/0000~000Q~s0~.
75 CJ 1982
Elwvicr
Scientific
Publishing
Company
cells were injected into mice (10 groups of 5). Seven days after inoculation, all groups but one (to be used as a control for both species) were treated every other day for 3 weeks with gallium nitrate solutions containing various concentrations of sodium citrate. The amount of gallium thus administered totalled in all cases 8 mg kg* of body weight, whereas that of sodium citrate ranged from 0 to 30.5 mg kg”’ of body weight. After spontaneous death of the animals, tumour-affected tissues as well as healthy organs (lung, heart, liver, spleen and kidney) were escised. The weight ranges of the various organs are shown in Table 1. All samples were kept frozen until treatment could be started. The organic samples were wet-ashed in concentrated nitric acid (65%, Merck Suprapur). Depending on the weight of the sample, lo-50 cm’ of acid was used. The mixtures were evaporated over a small flame until a clear solution was obtained on cooling. No filtration was found to be necessary_ The procedure resulted in complete destruction of the organic material and conversion of gallium to its nitrate_ The volume of the solutions thus obtained was 2-3 nitric acid and subcm3. These were made up to 10 cm3 with concentrated sequently diluted (1 + ‘9) with double-distilled water. Three identical aliquots of each were finally taken for determining gallium_ There is no serious risk of external contamination by gallium. Nevertheless, thorough rinsing of all g!assware with high-purity 1 M nitric acid was effected prior to use, and manipulations of the samples were minimized. Analyses were carried out by means of the three spectroscopic techniques mentioned after preliminary experiments for optimizing the working conditions for each. 411 measurements were carried out in triplicate and averaged. TGS
Atomic
absorption
spectrometry
(a.a.s.)
Measurements were made with a Perkin-Elmer model 430 atomic absorption spectrometer and a carbon furnace, connected to a model 56 recorder. The furnace tubes were of pyrolytic graphite to allow a larger number of injections (ca. 100) with each tube. High-purity argon was used to purge the furnace at 900 ml min-’ in an interrupted flow mode. Although preliminary results showed that background correction was
TABLE
1
Range of organ weights
Organ
Lung Heart Liver
in rats. and mice
Weight (g) --_---_._-
~-
RatS
Mice
1.3-2.1 1.1-1.6 8.5-l 1.0
0.2-0.4 0.1-0.1s 1.1-1.5
Organ
Spleen Kidney Tumour
Weight (g) Rats
Mice
0.8-1.6 1.9-2.4 2.1-8.5
0.24.4 0.1*.25 0.4-1.4
superfluous during the atomization stage, it was decided to apply it merely to minimize recorder scale deflection caused by smoke formation during the thermal decomposition phase. Eppendorf pipettes were used to inject 10 ~1
of
solution
furnace. The main operating parameters nm; lamp current, 15 mA; furnace conditions, lOO”C, 5 s, rate 2; 8OO”C, 5 s, rate 2; 25OO”C, 7 s, rate 0 (gas interrupt). Calibration graphs were constructed from the peak heights obtained from solutions containing 10, 50, 200, 1000 and 1500 ng Ga ml-’ in 10% nitric acid. Differences in salt content of the various organs generally, and sometimes remarkably (as in the case of liver .samplcs), affected the reliability of measurements and thus led to erratic results. This could be ascertained by plotting calibration graphs for gallium in solutions obtained from different organs and comparing them with the graphs pertaining to solutions containing only nitric acid and gallium nitrate. In all the cases investigated. the slope of the calibration plots was significantly different to that obtained with gallium nitrate and nitric acid. Therefore it was concluded that for each type of sample it is necessary to resort to the standard addition method, which proved successful for the elimination of some systematic errors. An effective standard reference material, however, would afford more consistent data. adopted
were:
manually
into
wavelength,
the
287.4
Arc emission spectrography :\ PGS-2 plane-grating spectrograph
(VEB Carl Zeiss Jena, 650 grooves mm-i, blazed at 5” 35’. Kbert mounting, 20-pm entrance slit, intermediate diaphragm 3.2) was used with the arc emission source, which was equipped with an arc generator (Optica, Milan). The procedure adopted was based on the standard addition method. because matris effects were noticeable, though to a lesser extent than with a.a.s. A lo-cm’ aliquot of sample solution was first added with an equal volume of a solution containing 10% sodium sulphate and 1% sulphuric acid as well as various aliquots of the standard gallium solutions. The mixtures thus obtained were dried under ix. irradiation and calcined at 350°C for 3 h. The residues were accurately homogenized and introduced in the crater of graphite electrodes (Ringsdorff RW 0 52 anode cup), and arced at 8 A for 35 s, to ensure complete consumption of the added sample. The gallium line at 294.4 nm was preferred because it was practically free from interference from other elements and reflected concentration variations reliably. Spectra were photographed on Kodak Spectrum Analysis No. 1 plates. Plates were developed in a Kodak solution D-19 for 4 min at 20 ? O.l”C. The blackening values were measured by a microdensitometer MD-100 (Jenoptik Jena, GDR), capable of evaluating blackening as great as 3.2 with an error of less than 2%. Correction for background required the preliminary elaboration of a sensitometric struction of standard addition
curve, which was also calibration graphs.
necessary
for the con-
228
Hollow-cathode
emission
spectrography
The instrumentation employed was a vacuum spectrograph SPV lm/800 equipped with a l-m radius concave grating with 1200 grooves mm-*, blazed at 5” 52’, in a Paschen-Runge mounting, with a 30+m entrance slit, a HVG 2 electric source and a hollow-cathode lamp, all supplied by WV-Prtiisionsmessgetitc GmblI (8031 IIechendorf/Pilsensee, \V. Germany). High-purity argon at 550 Pa was used as the filler gas, because both its ionization potential and atomic mass are sufficiently high to assure an appreciable sputtering of cathodic material. The lamp current, was 400 mA, and the voltage 275290 V. A 4-min exposure of SA No. 1 emulsion was used for photographing
the spectra, supported on film. Films were processed under the same con-
ditions previously mentioned and the blackening at. the 417.2-nm gallium line was read with the MD-100 microdensitometer. The most suitable material for preparing holIow cathodes was stainless steel, from the point of view of both excitation stability and analysis sensitivity. Materials such as graphite and aluminium were also tested, but for various reasons were considered inconvenient and therefore not used further_ Preliminary tests were carried out to ascertain whether matrix effects were relevant or not.. Results showed that calibration graphs obtained by the standard addition method in the presence of different matrices gave no appreciable variation in slope and intercept. so that one calibration graph would suffice for all analyses. The nitric acid solutions obtained from the mineralization of the organic samples were introduced as 400+1 aliquots by means of a micropipette into the hollow cathode held vertically with its open end up. The cathodes were dried under i-r. irradiation and calcined at 500°C for 30 min. The 417.2-nm gallium line was chosen as the others were either not sufficiently intense or were masked by lines emitted by the cathodic material. Visual inspection after the discharge of the inner part of the electrode revealed that uniform sputtering occurred over the walls of the back half of the cathode bore, including the surface at the closed end. Under the experimental conditions of current intensity and gas pressure adopted for the discharge, it is in fact the lower part of the inner surface that is expected to contribute more effectively to the sputtering process. Therefore, a regular and efficacious progress of the discharge can be assumed for the present determination. RESULTS AND DISCUSSION
Typical gallium concentrations determined in the organic samples are set. out in Table 2. These values are representative of those groups of rodents (one for rats and one for mice) which showed the longest survival time. In the case of rats this corresponded to gallium formulations containing the largest amount of sodium citrate (equal to 30.5 mg kg-l body weight), whereas for mice the most suitable concentration of sodium citrate was found to be 7.5 mg kg-* _ The meaning of these data, however, will not be
229 TABLE
2
Average concentration Organ
of gallium found in the various organs (~g g- ‘)
Atomic absorption spectrometry ---I Rat
Lung Heart Liver Spleen Kidney Tumour
0.36 0.17 3.5 1.6 1.8 1.5 ~
Arc emission spectrography -Rat .----_-._-
Mouse _____ 0.4 1 0.09 2.09 0.78 1.48 0.96 __--- -
-
0.38 0.18 4 .o 1.7 2.0 1.7
.-Mouse
--Hollow-cathode emission spectrography .-Rat -
0.44 0.10 2.2 0.90 1.70 1.03 _.-
0.36 0.18 3.6 1.6 2.0 1.5 ..-
-
-.-
hlousc ---. 0.39 0.07 2.2 0.85 I.45 1.03
examined in this paper, because the main purpose here is to elucidate the applicability of spectroscopic methods to the determination of gallium in biological material. In order to make a reliable comparison of the analytical capabilities characterizing the three techniques, all determinations were repeated many times (never less than 10 measurements for each sample), and the results averaged. The parameters which allow an overall evaluation of each method are given in ‘Fabic 3. As far as sensitivity is concerned, in a.a.s. this is usually the concentration corresponding to 1% absorption and not simply the slope of the linear plot of the analytical signal as a function of concentration. Nevertheless, in this study it ‘cvas decided, for the purpose of comparing results, to calculate the sensitivity of the a.a.s. determinations on the latter basis, as done for the
TABLE
3
Characteristics samples hfcthod --Atomic absorption Arc emission Hollow-cathode emission
of spectroscopic
techniques
__ Uctcction limit“ (ppm)
__-_...__ Precisionb (6)
0.005
-I
0.1 0.01
9 2
for the determination
of gallium in biological _-
-..--
_.-._.
Retative sensitivityE
hlatrix t?ffect
98
0.55
High
10.1 102
0.20 1 .OO
Intermediate Low
hlean
recovery (S) _--___---
---
of variation, as calcutatcd on the basis of 10 measurements “3~ background. “Coefficient for a.a.s. and 12 for the other two m&hods. cSopc of calibration graph divided by standard deviation.
230
other two spectroscopic methods. The procedure described by Grant [7] was adopted in order to normalize sensitivities espressed with different units of the analytical signal. A comparison of the sensitivities pertaining to different analytical methods is in fact not directly possible because the signaf is
espressed by units typicaf for the technique considered (absorbance for a.a.s., blackening units for spectrography). Any analytical procedure can, however,
be essentially characterized by precision and sensitivity, both quantities being related to the particular units adopted. Therefore, the quantity obtained by dividing sensitivity by the standard deviation for a given method gives results independent of any particular system of units and, as a consequence, allows a meaningful comparison of data referring to different techniques. The accuracy of the methods was tested by doping, with 0.2 and 1 mg 1-l. the sample solutions obtained after wet ashing of biological material from rodents not treated with gallium and therefore free from detectable levels of this element. The corresponding mean recoveries are reported in Table 3.
Two principal considerations can be made on the basis of the results obtamed in this study. All the spectroscopic methods tested afforded reliable measurements and proved to be suitable for determining gallium in biological samples. Hollow-cathode emission spectrography appears to be superior to the other techniques insofar as it is relatively free from matrix interferences. Sputtering sources such as this are also characterized by high reproducibility and favourabIe detection limits [S, 91, although the latter are not as good as in a.a.s. Generally speaking, emission spectrographic techniques require a greater number of manipulations, this being a not negligible drawback and usually prolonging the time necessary for the analysis. In the case of hollowcathode emission spectrography, however, the possibility of utilizing a single calibration plot for all determinations (as a consequence of the freedom
from matrix effects) largely counterbaiances sample manipulation.
the disadvantages of longer
Comparison of the different features of the three techniques indicates that hollow-cathode emission spectrography is the most suitable, even though a.a.s. presents some remarkable advantages insofar as rapidity of analysis and detection limits are concerned. From this point of view it must be acknowledged that spectrographic methods cannot compete with a.a.s. because such tedious and time-consuming steps are involved as processing of emulsions and evaluation of spectrograms. This aspect, however, does not really affect
the intrinsic advantages of hollow-cathode emission spectrography which depend essentially on the discharge properties and not on how the signal is detected. It is conceivable, therefore, that direct-reading spectrometers would greatly improve the speed of determination for the hollow-cathode emission source without altering the basic features of this source. This fact
should be borne in mind while considering the analysis times which are, for a series of 12 determination, of the order of 2, 4 and 4 h for a.a.s., arc emission spectrography and hollow-cathode emission spectrography, respectively,
23 1
(12 determinations of esposures which
were chosen because they correspond can be taken on the same film or plate).
This paper was September 1981.
presented
in part
at
the
XXII
to
CSI/Sth
the
number
IC?\S.
Tokyo,
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