Comparative Study of Atomic Absorption Spectrometry and

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					Journal of Analytical Toxicology,Vol. 23, November/December1999

Determination of Germanium in Human Specimens:
ComparativeStudyof AtomicAbsorptionSpectrometry
and Microwave-InducedPlasmaMassSpectrometry
AtsukoShinohara, Momoko Chiba, and Yutaka Inaba
Departmentof Epidemiologyand EnvironmentalHealth,Juntendo UniversitySchoolof Medicine, Tokyo 173-842l, Japan

I Abstract I                                                                          6 deaths, have been reported from 1982 to 1988 (3). These hap-
                                                                                      penings were associatedwith Ge-containingdietarysupplements.
The determination methods of germanium (Ge) in biological                             Ge was believedto have health benefits because some medicinal
specimens such as blood plasma, erythrocytes, urine, hair, nail, and                  plants have high Ge content and antitumor or immunomodula-
other organs were established using graphite furnace atomic                           tion properties of several organogermanium compounds have
absorption spectrometry (GFAAS) and microwave-induced plasma                          been reported (4,5). Ge-containingtablets or elixirshave not been
mass spectrometry (MIP-MS). The detection limits of Ge standard
                                                                                      sold in general drug stores in Japan since an administrativeguid-
solution were 3 ng/mL with GFAAS and 0.05 ng/mL with MIP-MS.
The detection limits in organ samples depended on the type of
                                                                                      ance in 1988. However,several cases of Ge-inducedrenal toxicity,
samples and sampling amounts: 3-30 ng/g by GFAASand                                   neurotoxicity,and renal failure of diabetics have been reported in
0.05-0.5 ng/g by MIP-MS. The sensitivity of GFAAS was lower than                      subsequent years (6,7). Most of the Ge intoxication was induced
that of MIP-MS; however, it was adequate for determining Ge                           by the intake of inorganic germanium oxide (GeO2). Renal dys-
concentrations in specimens from patients who had ingested Ge.                        function was also induced with organogermanium compounds
Samples were digested by a simple wet-ashing procedure using nitric                   such as germanium lactate citrate (8). Until recently, another
acid and perchloric acid. To avoid the interfering effects of coexisting              organic Ge compound, carboxyethylgermaniumsesquioxide(Ge-
elements and perchloric acid residue, an extraction method using                      132), was used as a drug for research and was administered to
organic solvent was tried. When using MIP-MS, extraction was not                      patients in Japan suffering from cancer or rheumatism. In addi-
necessary; however, both dilution and addition of an internal standard                tion, 3-oxygermylpropionicacid polymer (propagermanium)has
were needed. Special attention was required for iron-rich samples
                                                                                      been used in Japan since 1994 for the treatment of chronic hep-
because a molecular ion of S6Fe160was observed at m/z 72 where
Z2Ge was monitored. The results of Ge concentrations in human
                                                                                      atitis B because of its modulating action on host responses (9,10).
samples obtained by these methods agreed well. Interfering effects of                    A sensitive and selective method for Ge determination in
perchloric acid, which was used for digestion and which remained in                   human specimens is necessaryfor checking the accumulation of
samples, were observed in both methods. Hair and nail samples from                    Ge in the body to avoid intoxication and for checking adverse
people who had ingested Ge were useful for monitoring Ge in the                       effects of Ge-containing compounds or drugs. Organ samples
body. Hair samples were useful for determining past exposure to Ge                    obtained by biopsyor by autopsyprovidedirect information about
when the distribution patterns from the scalp to the end of the strand                Ge concentrations; however,these samples are available only in
were analyzed. In control subjects, Ge concentrations in the listed                   special cases. Bloodand urine are common samples for diagnostic
specimens and organs were lower than 0.1 pg/g or mL, and these low                    purposes and are considered to be good indicators reflecting the
levels of Ge were able to be determined by MIP-MS in combination                      present state of Ge in the body. Hair and nail samples can be
with the extraction method.                                                           obtained painlessly and are expected to indicate recent or past
                                                                                      exposure to Ge. These biological specimens contain alkali and
                                                                                      alkaline earth elements at high concentrations, and the amount
                                                                                      of sample availablefor analysis is often limited.
                                                                                         Various techniques of spectrophotometry, atomic absorption
 Introduction                                                                         spectrometry (AAS),and other methods have been reported for
                                                                                      Ge determination (11-15). High-frequency plasma mass spec-
  Germanium (Ge) is used mainly in industrial fieldsand is found                      trometry, such as inductivelycoupled plasma mass spectrometry
ubiquitously in soil, water, plants, foods, and biomaterials (1).                      (ICP-MS) and microwave-induced plasma mass spectrometry
Although the toxicity of Ge is low compared with other heavy                           (MIP-MS) can allow sensitive and multielement analyses.
metals, there havebeen more than 30 reported human casesof Ge                          Graphite furnace atomic absorptionspectrometry (GFAAS)is also
intoxication (2). In Japan, 23 cases of Ge intoxication, including                    available and has the advantage of being widely used and

                                Reproduction(photocopying) editorialcontentof thisjournalis prohibitedwithoutpublisher'spermission.                 625
                                                                                Journal of Analytical Toxicology,Vol. 23, November/December1999

common. ICP-MSand MIP-MSequipment is relativelyexpensive              1987. Specimens from Patient A were taken during the autopsy
but has recently become available in many laboratories.With this      and obtained through a physician 14 months after the patient's
in mind, we developed suitable analytical conditions for deter-       death; specimens were stored at -80~ Patient B, a 60-year-old
mining Ge in biologicalmaterials using two methods: GFAASand          male, was aliveand had been diagnosedwith diabetes mellitus. He
MIP-MS. These methods were applied to human specimens from            had a past history of GeO2 intake over approximately 10 years.
patients who had ingested Ge compounds and to a control group.        Patient C, a 48-year-oldfemale,was aliveand had ingested Ge-132
                                                                      for several months in the past. Patient D, a 68-year-old male, had
                                                                      taken organogermanium to maintain a healthy body and had no
                                                                      symptoms of Ge toxicity.In order to obtainsamples from patients,
Experimental                                                          informed consent and approval according to the criteria of an
                                                                      intramural ethical committee were obtained by individualphysi-
Apparatus                                                             cians.
  GFAAS (Polarized-Zeeman atomic absorption spectrometer                The reagents, AAS-special-gradeHNO3,HCIO4,and HC1,analyt-
Z-6100, Hitachi, Tokyo, Japan) and MIP-MS (P-7000, Hitachi)           ical-grade CCI4,and Ge standard solution (1000 IJg/mL)for AAS
were used. The operating conditions are listed in Table I.            were purchased from Wako Pure Chemical Industries (Osaka,
Acetylene-flameAAS (Polarized-Zeemanatomic absorption spec-           Japan). Deionized water through Milli-XQ (Millipore, Tokyo,
trometer Z-6100, Hitachi) was used for determining Na, K, Ca,         Japan) with relative resistance of 18.3 M~ was used.
and Fe concentrations.
Materials                                                               Ten to 100 mg of hair, nail, and organ sample, 200-400 pL of
  Nail, hair, blood, urine, and organ specimens from controls and     plasma and erythrocytes, and 1 mL of urine sample were weighed
patients were analyzed. Controls had no history of ingesting Ge       precisely in demetalolyzed Pyrex glass tubes. Hair and nail
compounds. Nail, hair, blood, and urine were obtained from two        samples were washed with deionized water to remove external
laboratory volunteers, and nail, hair, and organ samples were         contamination and were air driedbefore weighing. These samples
obtained from 12 bodies donated for medical anatomy dissection.       were heated at 120-140~ with 2 mL HN03and 0.2 mL HCIO4on
The other specimens were from four patients who had ingested          a block bath (Advantec,Tokyo,Japan) to digest organic compo-
Ge compounds in the past. Patient A, a 58-year-oldmale, ingested      nents, then diluted with 0.5% HNO3. A series of standard solu-
GeO2 for 1-2 years and died from renal failure and myopathy in        tions was prepared from the Gestandard solution by dilution with
                                                                      0.5% HNO3.
  Table I. Operating Conditions of GFAAS and MIP-MS
  for Ge Determination
  GFAAS(HitachiZ-6100)                                                Results
      Resonancesource: Hollow-cathode lamp
          Lamp current: 12.5 mA                                       GFAAS
  Analytical wavelength: 265.2 nm                                       Four types of standard series as shown in Figure 1 were tried.
  Background correction: Polarized-Zeeman
                                                                      The calibration line of the Ge standard solution prepared with
              Slitwidth: 0.4 nm
                                                                      0.5% HNO3 was linear from 10 ng/mL to 1 IJg/mL when the
  Graphite furnaceoperation                                           sample volume injected into the furnace was fixed at 10 ]JL
        Atomization tube: Untreatedgraphitetube                       (Figure 1A, line a). Ge standard solution showed a straight line
        Injection volume: 10 pL                                       over the range up to 2 !Jg/mL.Asa matrix modifier,addition of Ni
               Carrier gas: Ar                                        was tried. Addition of Ni(NO3)2solution increased the sensitivity
  Heating program        Temp(~          Time (s) Gas flow (mt/min)   dose dependently.The optimumamounts of Ni addition were 1 IJg
  Dry                        80~120        30           200           or more. In the present study, 10 pL of 100 ]ag/mLNi solution was
  Char                          700        30           200           injected into the furnace together with 10 ]JL of the sample solu-
  Atomize                      2700        10             0           tion. The slope of the standard curve was about three times higher
  Clean                        2900         4           2O0           with Ni modifier than that obtained without modifier. Detection
                                                                      limit of Ge under the present conditions was 3 ng/mL, which
  MIP-MS (HitachiP-7000)
                                                                      corresponded to 0.03 ng.
                   Power:    1.3 kW                                     A series of standard solutions were digested under the same
         Plasmagas flow:     13 L/min                                 conditions as sample digestion. When 1 mL of standard solution
         Carrier gas flow:   1.3 L/min                                was heated with 2 mL HNO3 and 0.2 mL HCIO4at 120-140~ for
          Sampling cone:     Pt-made                                  2-3 h and the remaining residue was diluted to 1 mL with 0.5%
                                                                      HNO3, the calibration line was linear, but the slope was much
        cooling chamber:     4oc
                                                                      lower than that without digestion (Figure 1A, line b). A loss of Ge
      Scanning conditions:   Dwell time:50 ms
                             Numberof sweeps:3                        during the digestion process was observed when the remaining
                             Sampleflow:0.24 mL/min                   acid was volatized to dryness at 200~ The lost percentage of Ge
                                                                      varied from 25 to 75% depending on the tube position on the

Journal of Analytical Toxicology, Vol. 23, November/December 1999

heating block. In the following experiments, temperature was           10% under the present conditions. Addition of HCIO4to the cali-
kept at 140~ for 3 h in order to have HC104remain in the tube,         bration standard solutions decreased ion counts of Ge at m/z 72
and the remaining residue diluted with 0.5% HNO3was applied to         depending on the amounts added, and ion counts reached 75%
GFAAS. It was found that an addition of HCIO4to Ge standard            when HC104volume was 10%. This suggests that the decrease in
without heating process showed a dose-dependent decrease of            ion counts of digested standards was due to the remaining HCIO4.
absorption, suggesting the interference from the remaining             When the standard solutions were digested with biological sam-
HCIO4. When a series of calibration standard solutions were            ples, ion counts were decreased depending on the types and
digested with 0.5 mL of urine or with 50 mg of hair from a con-        amounts of samples. With 10 pL or 10 mg of plasma, erythro-
trol, calibration curveswere flat (Figure ]A, lines c and d). The Ge   cytes, urine, hair, or nail sample as the original amount before
absorption was also lowered when the Ge standard solutions were        digestion, the standard curves were linear, and their slopes were
mixed with digested urine or hair samples. These results indi-         from 93.1 to 102.9% of digested standard without biologicalspec-
cated the interfering effects of coexisting elements in these sam-     imens. In the case of plasma and urine samples, the ion counts of
ples. To avoid interfering effects of matrix elements and/or           Ge at rn/z 72 decreased with increased amounts indicating the
remaining perchloric acid, an extraction method was applied.           interfering effects of matrix elements (Figure 2A). In the case of
Digested standard solution with or without biological materials        erythrocytes, the ion counts decreased until the addition of 50 pL;
was extracted in 1 mL CC14under 8N HCI and then re-extracted           however,they increased again with the addition of 100 pL (Figure
in 1 mL deionized water according to the methods reported by
Mino et al. (12)with slight modifications.The absorbance showed          Table II. Ge Concentrations in Human Samples
a linear relationship with Ge concentration (Figure 1B) when the         Determined by GFAAS and MIP-MS
added amounts of urine, hair, nail, plasma, or erythrocytes were                                                     Ge (pg/mLor g)*
1.0 mL, 50 rag, 20 rag, 0.1 mL, or 0.1 mL, respectively,that is, the
original amounts beforedigestion. The slope of the digested stan-        Sample                        GFAAS            MIP-MSt           MIP-MS*
dard was the same as that of the standard without digestion. The
slopes of digestedstandards with biologicalspecimenswere in the          PatientA
                                                                         Pancreas                     2.4 •    0.9     2.78 • 0.38      2.81 • 0.51
range of 98 to 114% of the digested standards without biological
                                                                         M. iliopsoas                 2.4 •    0.7      2.54 • 0.75        2.50 • 0.87
specimens. Ge contents in samples were calculated using a
                                                                         M. quadriceps femoris        5.1 •    1.1     4.69 • 0.43       4.62 • 0.49
matrix-matched calibration curve that contained corresponding            M. pectoralis                4.7 •    1.2     4.54 • 1.20       4.96 • 1.19
amounts of the specimen obtained from controls.
  Removalefficiencyof major elements in these specimens using            PatientB
                                                                         Serum                         < 0.05          0.019 • 0.003    0.018 • 0.002
this extraction method was checked by flame AAS.When 1.0 mL
                                                                         Urine                       0.25 • 0.06       0.226 • 0.012    0.235 • 0.008
of urine was digested, Na and K concentrations in the aqueous
                                                                         Hand nail                   12.6+1.9         12.97•           12.74•
phase beforeextraction were 5312 and 752 pg/mL, and those after          Foot nail                   16.9•            16.62+7.59       15.86+8.02
extraction were 24 and 3 pg/mL, respectively, indicating that            Hair                        53.1 • 5.6        56.72 • 13.43    59.62 • 13.75
more than 99.5% of Na and K were removed. When 0.1 mL of
                                                                         * Mean • SD (n= 3).
plasma or erythmcyte was digested, the concentration of Na in            + With dilution only.
the plasma sample and the concentrations of K and Fe in the ery-         * With dilution and extraction.
throcyte sample in aqueous phase were decreased from 278, 243,
and 98 pg/mL to 1.5, 0.3, and 0.1 pg/mL, respectively,by extrac-
                                                                         Table III. Ge Concentrations in Specimens from Patients
tion. The removal efficiency was more than 99.4%. Similarly,             Detemined by MIP-MS with Dilution
more than 98.5% of Ca was able to be removed from nail and hair
samples by this extraction method.                                                                                   Ge (pg/mLor g)*
                                                                         Sample                     PatientA            PatientC        PatientD
  Ge has fivestable isotopes ofm/z 70, 72, 73, 74, and 76 with nat-      Plasma                            -          0.001 • 0.001
ural abundances of 21.2, 27.7, 7.7, 35.9, and 7.4%, respectively.        Erythrocytes                      -          0.021 • 0.007
Calibration standard curves showed linearity in the range of 0.5 to
500 ng/mL. Within these isotopes, m/z 70 coincides with one of           Hair                     71.05 _+6.31        4.89 + 0.53      10.96 + 0.91
the isotopes of zinc (Zn), and rn/z 74 and 76 coincide with iso-         Nail (hand)                16.90 • 4.88             0.02 t    55.14 + 9.40
                                                                         Nail (foot)              0.200 • 0.069       53.48 • 16.67
topes of selenium (Se). Therefore, ion counts at m/z 72 and 73
were preferentiallyused for Ge determination for Zn- and Se-con-
                                                                         Spleen                   49.74 + 2.93              -
taining biologicalsamples. The detection limits of Ge were 0.05
                                                                         Kidney                    6.15 • 1.08              -
and 0.2 ng/mL at m/z 72 and 73, respectively,when 3 a definition         Lung                      3.93 • 1.66              -
was used.                                                                Stomach                   3.90 • 0.65              -
  The effects of digestion and coexisting elements in the sample         Heart                     3.28 • 0.85              -
on Ge determination were also checked. Digestedstandard solu-            Muscle intercostal         1.98 • 1.27
tions without biologicalsamples showed a straight line; however,
                                                                         * Mean • SD (n=3).
the slope was 74.5% of that without digestion. The volume of             * Mean (n=2).
HCIO4 in digested standard solutions was estimated to be about

                                                                                                                      Journal of Analytical Toxicology, Vol. 23, November/December 1999

2A). A correction based on an internal standard was performed                                              inal amounts.
using gallium (Ga) with 10 ng/mL as the final concentration                                                  In the case of erythrocytes, the ion counts at rrgz 72 increased
because the concentrations of Ga in these biologicalsamples were                                           with sample volume, but those at m/z 74 did not, suggesting the
under the detection limit and the mass-to-charge ratios of Ga, 69                                          existence of molecular ions at m/z 72. The spectra of erythrocytes
and 71, were close to the mass-to-charge ratios of Ge. Biological                                          and other samples of a control subject were measured from ra/z
samples contained a lot of chloride, and the molecular ion                                                 71 to 76. As shown in Figure 3, the erythrocyte showed a peak at
3~Cl1602with ra/z 69 was easily formed; therefore, the ion counts                                          rrUz72 but did not indicate any clear peak at ra/z 74 (Figure 3, line
of ~lGa were used. The slopes of calibration lines obtained with                                           b). With other specimens such as plasma, urine and hair, a similar
standard solutions digested with and without biological samples                                            peak was not observed (Figure 3, lines c-e). Erythrocytes contain
were in the range of 98.5 to 101.8% of the original standard solu-                                         much higher concentrations of iron than the other specimens.
tion. The matrix effects on the ion counts were negated by                                                 The spectra of 100-pg/mL Fe solution indicated a clear peak at
internal standard correction when the added amounts of biolog-                                             m/z 72. The solutions of other biologicallymajor elements, Na, K,
ical samples were 50 pL or ng or less (Figure 2B).                                                         Ca, Zn, and Mg, did not indicate a matching peak, suggesting that
  Matrix effects become a potential problem when samples that                                              the peak at m/z 72 was associatedwith iron (Figure 3).
contain very low levelsof Ge must be large in measuring solution.                                            These results indicate that the internal standard correction is
The extraction method mentioned in the GFAASsection was                                                    necessary for all samples and that the extraction method is useful
examined to decrease the effect of coexisting elements. The                                                for samples that contained low levels of Ge. The rrgz 72 is prefer-
recovery rates of 50 ng/mL Ge from standard solutions prepared                                             entially used for Ge determination, but rrUz 73 is recommended
with 0.5% HNO3 to final aqueous phase were 90.8 • 1.5%. The                                                when iron-rich samples are measured without extraction.
extraction efficienciesof standard solutions digested with biolog-
ical specimens varied between 93 and 103% of standard solution                                             Determination of Ge in patients and control subjects
only when 1 mL measuring solution contained ~ 1 mL of plasma                                                Ge concentrations in specimens from Patients A and B were
or erythrocytes, ~ 2.5 mL of urine, or - 250 mg hair as their orig-                                        determined by both GFAASand MIP-MS.The values obtained by

                     OAI                                                A                                                                                     B
                                                                                                                                                           f-~6d   g

            j        0.4                                       b                  b                        ~
                                                                                                                                                                               a e w

                                                                                  C                        0
                                                                                  d                        ~   0.t
                                                                                                                                                                               9 o~..,

                                                                                                                                 i                  J
                                                                                                                                2oo                4oo                 6oo

                                             Ge (ng/mL)                                                                              Ge (ng/mL)
  Figure1. Calibration lines of Ge measured by GFAASwithout extraction (A) and with extraction (B). Identification: (a, @) standardsolution; (b, &) standard solu-
  tion subjected to the same procedure for the digestion of biological samples;and standard solution digested with (c, II) 50 mg hair, (d, [-]) 1 mL urine, (e, 9 0.1
  mL plasma, (f,/k) 0.1 mL erythrocytes, and (g,V) 20 mg nail.

                           2060o                                                  A              2.5
                o                                                                           m
                O                                                                           O
                O                                                                                                                                                       urine
                                                                                                 1,5                                                               9    plnma
                "6         1o000                                                            >                                                                           erythrocyte8
                                                                                            O     1                                                                e    nail
                g          5(x)o
                e.                                                                               0.5
                              0              i        i        i            i          i          0              i          L         L        L              I
                                   o    20       40       60       80           10o   120              0        20     40       60        80       11111     120

                                   Amounts of biological sample added                                 Amounts of biological sample added
                                                 (mg or pL/mL)                                                 (mg or pL/mL)

  Figure2. Effectof added amountsof sampleson ion counts at m/z 72 measuredby MIP-MS (A) and thosewith internal standardcorrection (B).The concentrations
  of Ge standard and Ga (internal standard)were fixed at 50 and 10 ng/mL, respectively.

Journal of Analytical Toxicology, Vol. 23, November/December 1999

GFAAS, by MIP-MS with only dilution, and by MIP-MS with                                     symptoms of Ge intoxication; however, exposure to Ge was
extraction agreed well, as shown in Table II. Ge in serum from                              thought to be remarkable because of evidence of high Ge concen-
Patient B was under the detection limit by GFAAS but was                                    trations in hair and nail (Table III).
detectable by MIP-MS.These results indicate that both methods                                 Ge concentrations in specimens from control subjects who had
are useful for Ge determination in biological specimens and that                            no history of specific Ge ingestion were very low; therefore, the
the sensitivity is higher in MIP-MS than GFAAS.Ge concentra-                                samples were measured by MIP-MSwith extraction. As shown in
tions in other specimens from Patient A and in specimens from                               Table IV, Ge concentrations were lower than 4 ng/mL (or g) in
Patients C and D were measured by MIP-MSwith dilution only                                  hair, nail, plasma, and urine from two controls. Ge concentra-
after digestion (Table III).                                                                tions in nail, kidney, spleen, liver, lung, and bone from autopsy
  The Ge content in hair was the highest in the samples from                                control samples were lower than 70 ng/g. The averageconcentra-
Patient A,who ingested GeO2and died from myopathy and acute                                 tions of Ge in autopsy samples were as follows: lung > kidney >
renal failure (16), as shown in Tables II and III. Among soft tis-                          liver, bone, spleen > nail. Gender differenceswere not clear, but
sues, the highest concentration of Ge was found in spleen, fol-                             average Ge concentrations in kidney were higher in females and
lowed in decending order by kidney, lung, stomach, heart, and                               those in bone were lower in females.
pancreas. Ge concentrations in muscles were within the same
order, where concentrations in the quadriceps femoris and pec-
toralis muscleswere about 5 IJg/g,and those in the iliopsoasand
intercostal muscles were about 2 IJg/g. Patient B had ingested                              Discussion
GeO2 for 10 years before stopping. Hair, nail, serum, and urine
samples were obtained 3 times at 2-3-month intervals. Table II                                The results of this work indicate that both GFAASand MIP-MS
shows the results of the first sampling. The concentrations of Ge                           were useful for Ge determination in biological specimens. The
in serum from Patient B were 0.019 + 0.003 and 0.010 • 0.005                                detection limits of calibration standard solution were 3 ng/mL
I~g/mLat the second and third samplings, respectively. Ge con-                              with GFAASand 0.05 ng/mL with MIP-MS.The detection limits
centrations in urine were 0.039 and 0.051 IJg/mL,respectively,at                            in organ samples depended on the types of specimens and sam-
the second and third samplings. Ge concentrations in nail and                               pling amounts; when 1.0 mL of plasma, erythrocytes, or urine;
hair were not clearly changed during this period.                                           0.25 g of hair; or 0.1 g of nail or other organ was digested and
  Patient C had ingested Ge-132 but stopped the ingestion several                           back-extracted in I mL deionized water, detection limits in sam-
months before the collection of blood, nail, and hair. As shown in                          ples were between 3 and 30 ng/g by GFAASand were between 0.05
Table III, Ge concentrations in plasma and in nails were very low.                          and 0.5 ng/g by MIP-MS.In MIP-MSwithout extraction, digested
The concentration of Ge in hair sample of about 10 cm in length                             must be diluted at least 20 times from the original weight of sam-
from the scalp from Patient C was 4.89 + 0.53 lJg/g. This hair                              ples and internal standard correction must be adopted to avoid
sample was cut into 1-cm pieces, and Ge concentration in each                               interfering effects of coexisting elements and remaining HCIO4.
piece was determined. Ge concentrations were different depend-                              The detection limits of MIP-MSwithout extraction were 1 ng/g.
ing on the piece, being higher at the end of the strand, then                                 The effectsof coexistingelements and HCIO4were thought to be
decreasing to levels under the detection limit at distances of less                         matrix interference, decreased sensitivitybecause of the sample's
than about 3 cm from the scalp (Figure 4). Patient D had no                                 having a greater viscosity than the standard solutions prepared

  Table IV. Ge Concentrations in Specimens from Volunteers and from Bodies for Anatomical Dissection Regarded
  as Control Subjects*

                                    Nail          Plasma               RBC      Urine         Hair       Kidney       Spleen Liver       Lung        Bone
   Subject        Gender           (ng/g)         (ng/mL)              (ng/mL) (ng/mL)       (ng/g)         (ng/g)       (ng/g)         (ng/g)
                                                                                                                                   (ng/g)           (ng/g)

    1                F            3.7 + 2.4       0.2 _+0.2        0.1 + 0.1    0.1+ 0.1    0.1+ 0.1
    2                F            3.8 + 2.2       0.7 + 0.5         < 0.05      0.4 + 0.6   2.7 + 0.8
    3                F               8.5                                                                 60.53        3.38      8.92       40.00     6.04
    4                F              16.8                                                                 20.97        23.93     9.56       59.67     6.19
    5                F              15.3                                                                 26.75        6.59       5.86      65.75     0.91
    6                F               5.5                                                                 41.61       17.88      6.81       20.97     0.00
    7                F               5.8                                                                 12.56       11.27     25.32       33.85     1.86
    8                F               2.9                                                                 26.25       13.33     29.86        7.49     0.00
    9                M               1.1                                                                  7.82        0.01     14.29       55.30    16.11
   10                M               1.7                                                                  9.84         9.34    16.38        39.22   18.32
   11                M             23.8                                                                  15.96        5.88     17.62       17.71    64.32
   12                M               3.6                                                                 29.17       22.38      7.01        NDt      6.63
   13                M                 0.6                                                               18.49       22.22     16.36       16.87    13.76
   14                M               3.8                                                                  2.27       16.59     27.03       51.52    57.02
   * Subjects 1 and 2: mean + SD, n = 3. Subjects 3-14: mean, n = 2.
   ~" Not determined.

                                                                                                Journal of Analytical Toxicology, Vol. 23, November/December 1999

with 0.5% HNO3and a decreasedsensitivitybecause of high salt                               of major matrix elements was very high, more than 98.5%.
contents. In GFAAS,HC104 may form a chloride of Ge that is                                   The concentrations of Ge in hair and nail were high in all
easily volatilized in the furnace. The concentration of Fe in                              patients, but very low in controls, suggesting that these speci-
sample must be checked in MIP-MSwithout extractionbecausea                                 mens were useful for biologicalmonitoring of Ge. Ge concentra-
molecular ion was observed at rn/z 72 with Fe solution at 10                               tions in fingernailsand toenailswere ofthe same order in Patients
lJg/mLor higher.A molecular ion of ~Fe]~ havingthe same m/z                                A, B, and D. In the case of Patient C, Ge concentrations in finger-
72 was thought to have formed. Among biological specimens,                                 nails were much lower than in hair, and Ge in toenails and in
attention must be paid to iron-rich samplessuch as erythrocytes,                           blood plasmawere very low, in the rangeof the controls. The dif-
liver, and spleen.                                                                         ferencesin Ge concentrations in these specimensmay reflectthe
  The sensitivity of GFAASwas lower than that of MIP-MS.                                   remaining amounts or chemical speciesof Ge in the body. Hair
However, it was adequate for determining Ge concentrations in                              analyses gave useful information about Ge intake in the past.
specimens from patients in order to check the Ge accumulation                              Patient C stopped ingesting Ge compound about six months
in the body.This technique is necessary for the extraction pro-                            before sample collection.Assumingthat hair growth rate was 1.1
cess; however, it is valuable for practical use because GFAAS                              cm/month, Ge was continually excreted in hair after Ge intake
equipment is widelyavailable in many laboratories.                                         was stopped. The excreted amount decreased gradually and
  MIP-MSwas more sensitivethan GFAASand was comparableto                                   reached undetectable levels during the three months prior to
ICP-MS (15). The extraction method was useful for determining                              sample collection.
Ge in specimenshavingvery low concentrationssuch as controls:                                Ge concentrations in specimens from controls were very
0.1--66 ng/g or ng/mL.This method would also be usefulfor iron-                            low, and their range was the same or a little lower compared
rich samples and for hard tissues such as bone and teeth, which                            with recent reports by ICP-MS (15). Schroeder and Balassa (1)
contain large amounts of Ca and P,becausethe removalefficiency                             reported Ge concentrations from 0.2 to 1.9 pg/mL in blood or
                                                                                                           urine from normal persons. However,we found
                                                                                                           one order of magnitude lower Ge amounts in
                                                                                     ] lmklls              blood and urine from three women who had no
                                                                                                           history of using Ge tablets or Ge-enrichedfoods.
                                                                                                           We also determined Ge concentrations in plasma
                                                                                                           from 70 healthy adults working in the same fac-
                                                                                                           tory to be 0.005 + 0.005 IJg/mL(17). The reasons
                                                                                                           for the differences in Ge concentration are
                                                                                              It           obscure, but we assumed that they would be
                                                                                              I            related to the differencesin Ge content in foods
                                         ~g                                                  J             and environments among countries or to the sen-
                                                                                                           sitivity and specificityof analytical methods used.
           70   71  72    73  74   75   78    77                70 71   72   73   74    75    76  7?
                                                                                                           Therapeutic effects of organogermanium are
                           m/z                                                  m/z
                                                                                                           currently being examined. These analytical
  Figure 3. Spectra of digested biological samples and standard solutions of Ge and other elements         methods are expectedto be useful for determining
  measured by MIP-MS in the rangefrom m/z 71 to 76. Identificaton: a, 10-ng/mL Ge standard solu-           the accumulation state of Ge in the body in order
  tion; b, erythrocytes (50 pL as original volume per milliliter measuring solution); c, 50 pL plasma;     to prevent patientsfrom sufferingfrom side effects
  d, 100 pL urine; e, 25 mg hair; f, 100 tJg/mL Fe; g, 100 pg/mL Ca; h, 100 pg/mL Mg; i, 100 pg/mL         of Ge-containingdrugs.
  Na; and j, 100 pg/mL K.

               12                                                                                      References

               10                                                                                       1. H.A. Schroeder and J.J. Balassa. Abnormal trace
                                                                                                           metal in man: germanium. ]. Chron. Dis. 20:
         .-.   8                                                                                           211-224 (1967).
                                                                                                        2. S.-T. Tao and P.M. Bolger. Hazard assessment of
                                                                                                           germanium supplements. Reg. Toxicol. PharmacoL
                                                                                                           25:211-219 (1997).
               4                                                                                        3. O. Wada and M. Nagahashi. Germanium intoxica-
                                                                                                           tion by foods for health. (In Japanese.) J. Jap. Med.
               2                                                                                           Assoc. 99:1929-1933 (1988).
                                                                                                        4. G. Falkson and H. Falkson. Phase 2 trial of spiroger-
               0                                                                                           manium for treatment of advanced breast cancer.
                        1     2      3    4     5   6     7              8      9                          Cancer Treat. Rep. 67:189-190 (1983).
                                    Distance from scalp (cm)                                            5. F. Suzuki, R. Brutkiewicz, and R. Pollard. Ability of
                                                                                                           sera from mice treated with Ge-132, an organic ger-
  Figure4. Distribution of Ge in the hair sample from Patient C. The hair sample was cut into 1-cm         manium compound, to inhibit experimental murine
  pieces from the scalp, and Ge concentrations were determined.                                            ascites tumors. Br. J. CancerS2:757-763 (1985).
                                                                                                        6. T. Asaka, E. Nitta, T. Makifuchi, Y. Shibazaki,

Journalof Analytical Toxicology,Vol. 23, November/December1999

    Y. Kitamura, H. Ohara, K. Matsushita, M. Takamori, Y. Takahashi,               electrothermal atomization. Chem. Pharm. Bull. 28" 2687-2691
    and A. Genda. Germanium intoxication with sensory ataxia.                      (1980).
    J. NeuroL 5cis. 130:220-223 (1995).                                      13.   C. Shleich and G. Henze. Trace analysis of germanium. Fresenius
 7. A. Takeuchi, N. Yoshizawa, S. Oshima, T. Kubota, Y. Oshikawa,                  J. Anal Chem. 338:140-144 (1990).
    Y. Akashi, T. Oda, H. Niwa, N. Imazeki, A. Seno, and Y. Fuse.            14.   K. Jin, Y. Shibata, and M. Morita. Determination of germanium
    Nephrotoxicity of germanium compounds: report of a case and                    species by hydride generation-inductively coupled argon mass spec-
    review of the literature. Nephrom. 60:436-442 (1992).                          trometry. Anal. Chem. 63:986-989 (1991).
 8. B. Hess, J. Raisin, Z. Zimmermann, F. Horber, S. Bajo,                   15.   J. Yoshinaga, M. Nakazawa, T. Suzuki, and M. Morita. Determination
    A. Wyttenbach, and P. Jaeger. Tubulointerstitial nephropathy                   of trace elements in human liver and kidney by inductively coupled
    persisting 20 months after discontinuation of chronic intake of                plasma mass spectrometry. Anal 5ci. 5:355-358 (1989).
    germanium lactate citrate. Am. J. Kidney Dis. 21 9548-552 (1993).        16.   M. Chiba, A. Shinohara, Y. Inaba, S. Nakayama, H. Rinno, and
 9. K. Asano, M. Yamano, K. Haruyama, E. Ilawa, K. Nakano,                         H. Koide. Two cases of patients taking germanium-containing food
    M. Kurono, and O. Wada. Influence of propagermanium (SK-818)                   stuffs. (In Japanese.)Juntendo Med. J. 36:406-410 (1990).
    on chemically induced renal lesions in rats. J. Toxicol. Sci. 19:        17.   M. Chiba, A. Shinohara, K. Matsushita, H. Watanabe, and Y. Inaba.
    131-143 (1994).                                                                 Indices of lead-exposure in blood and urine of lead-exposedworkers
10. Y. Ishiwata, E. Suzuki, S. Yokochi, T. Otsuka, F. Tasawa, H. Usuda,            and concentrations of major and trace elements and activities of
    and T. Mitani. Studieson the antivial activity of propgermanium with           SOD, GSH-Px and catalase in their blood. TohokuJ. Exp. Med. 178:
    immunostimulating action. Arzneimittelforschung 44:357-361                     49-62 (1996).
11. M. Anke and M. Glei. Handbook on Metals in Clinical and
    Analytical Chemistry, H.G. Seiler,A. Sigel, and H. Sigel, Eds. Dekker,
     New York, NY, 1993, pp 381-386.
12. Y. Mino, N. Ota, S. Sakao, and S. Shimomura. Determination of ger-                        Manuscript received September8, 1998;
     manium in medicinal plants by atomic absorption spectrometry with                         revision received November 25, 1998.


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