Quantitative X-Ray Diffraction Analysis of Urinary Ca

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					CLIN. CHEM. 34/2, 289-294 (1988)

QuantitativeX-Ray DiffractionAnalysisof UrinaryCalculi by Use of The Internal-Standard
Method and Reference IntensityRatios
M.A. E. Wandt’ andA..L Rodgers

The internal-standard method and the powder diftractometer                   Using the original internal standard technique (9), Chung
have been applied here to the quantitative determination   of             developed    a much simpler and faster “matrix-flushing”
urinary stone constituents by x-ray diffraction (XRD). Refer-             concept (10, 11), which involves use of reference intensity
ence intensity ratios determined for six stone substances                 ratios (12,13). In choosing corundum as the “flushing agent”
were used in the reduction of intensity data. Constituent                 (10), the mass fraction Xi of component J of the stone
concentrationscalculated for 21 stones were compared with                 powder mixture can be expressed in terms of the corundum
values obtained from an element-sensitive technique. We                   mass fraction Xc and the intensities of the corundum (113)
conclude that XRD analysis alone cannot be regarded as a                  reflection J and any reflection Ij (usually the strongest)  of
routine technique for the quantitative characterization of                compound J:
uroliths,but that semiquantitativeXRD analysis supplement-
ed by accurate quantitative elemental data is more suitable                  V     -

for the precise determination of true stone composition.                      “k1jIc

  Accurate    knowledge      of the qualitative  and quantitative         where the constant         k1 is the reference intensity   ratio
composition    of urinary calculi     is vital for understanding
their etiology and        for the development     of prophylactic
measures     (1). For the qualitative classification of calculi,          Rll:t,       =   ()so:so
x-ray diffraction (XRD) analysis has often been the method
of first choice (2). However, the implementation of quantita-
                                                                          determined    in a 50:50 (by wt) mixture of compound J and
tive XRD analysis has been impeded by the fact that most
                                                                          corundum C for the strongest reflection i.
stones are crystallographically          heterogeneous    and contain
                                                                             In the present study, reference       intensity ratios had to be
considerable     amounts of organic matter and loosely bound
                                                                          determined,    because only few such values have been pub-
water. In addition, the variable degrees of crystallinity            of
                                                                          lished for urinary-stone   constituents.
calculi components further complicate such analyses.
    While the small sample size, 0.1 to 0.3 mg, for the Debye-            Materials and Methods
 Scherrer method is advantageous, the Guinier film method
 features a much better resolution and allows simultaneous                Apparatus
 analysis of more than one sample. The Guinier goniometer                   We used a Philips automatic x-ray powder diffractometer
 (3) on the other hand, dispenses with recording the patterns             equipped  with PW 1050/70 vertical goniometer, PW 1390
 on film, but involves use of a counting tube. However,                   channel control, PW 1394 motor control, PW 8203 pen
 alignment     of this apparatus     is very complicated, which is        recorder, and PW 1395 programmer. Radiation was pro-
 why it is deemed unsuitable           for routine analysis (4). The      duced by a PW 2233/20 Cu normal focus tube set at 50 kV
 powder diffractometer         with Bragg-Brentano         geometry is    and 30 mA, which was allowed to stabilize for 30 mm before
 therefore probably the most widely applied instrument,                   measurements started.
 although at least 10 to 15 mg of sample is needed.
    The present study was undertaken            to examine and evalu-     Reagents
 ate quantitative      XRD procedures using powder diffracto-                Samples of calcium oxalate    monohydrate (whewellite),
 metry and to apply such an approach in the quantitative                  magnesium ammonium       phosphate hexahydrate (struvite)
 determination      of the constituent     concentrations in a group      (both from BDH), calcium phosphate hydroxide (hydroxyap-
 of urinary stones.                                                       atite) (Sigma), calcium monohydrogen phosphate dihydrate
     Of the five different experimental         techniques used today     (brushite) (Sarchem), and uric acid (Merck) were qualita-
  in the reduction of data in quantitative XRD analysis (5,6),            tively analyzed by x-ray powder diffraction to verify their
 the internal-standard       technique emerges as the only meth-          purity. Calcium oxalate dihydrate (weddellite) was prepared
 od that is largely independent of the sample under investi-              in our laboratory, and powder diffraction confirmed the
  gation. This method         has also been found to yield the            presence of only one hydrate. The internal standard used
  greatest precision in most cases (7). Although              it is not   was a-A1203 (corundum, BDH) with a mean particle size of
 totally free of inadequacies,         the technique is superior to       0.3 zm.
  other approaches      (8), because it accounts for any variation
  in the sample’s absorption coefficient.                                 Procedures
                                                                             A cryogenic grinding procedure (14) was developed to
                                                                          avoid structural changes during the pulverization     of stones.
  Department of Physical Chemistry, University of Cape Town,              Approximately 1 to 1.5 g of powdered sample were enclosed
Private Bag, ZA-7700 Rondebosch, Republic of South Africa.                in a specially machined PTFE cup together with a ceramic
   ‘Current address: Council for Scientific and Industrial Research,
National Accelerator Centre, Van de Graaff Group, P.O. Box 72,            ball of 1 cm diameter. A tight-fitting lid was held in position
ZA-7131Faure, R.S.A.                                                      with a stainless-steel clamp and the cup and contents were
 Received August 31, 1987; accepted November25, 1987.                     immersed in liquid nitrogen for about 20 to 30 mm. The cup

                                                                                                 CLINICAl..            34,No.2,1988 289
                                                                                                         CHEMISTRY, Vol.
was then mounted onto a “Mikro Dismembrator                    II” (B. the x-ray pattern that included the stronger reflections                of
Braun) and the grinding action was commenced. The low                  each component were step scanned (step size 0.01#{176}20, s         10
temperatures     permitted crushing to be continued for long           fixed time). We again processed             the patterns, using the
periods without the sample temperature rising above 0#{176}C “NBS*QUANT82”                 system (18). On a few occasions, program
and rendered the sample very brittle, thereby facilitating             “QXDA” (19) was used.
the grinding process. The low temperature also obviated
heat-induced changes.                                                  Results
   The resulting powders were mixed with about 30-40% (by                 Table 2 lists reference intensity ratios for the six sub-
wt) alpha-alumina standard         to yield aliquots of about 1 g.     stances measured-whewellite,            weddellite,   hydroxyapatite,
The cryogenic grinding and mixing procedure was then                   brushite, struvite, and uric acid-together             with published
repeated. Standards      (pure phase-alumina,     50% by wt) were      values. These constants were derived from peak-height
prepared in the same way. Samples were re-screened                     measurements, scaled to the strongest peak, and are thus
through 400-mesh (38 ian) screen to break up flakes that               directly comparable with listed values (20).
may have been oriented by the grinding.                                   Table 3 gives the results for 21 stones analyzed, which
   Powdered specimens were packed into standard Philips                agree well with results obtained from inductively                coupled
sample holders, a “back” or “rear” loading procedure being             plasma atomic emission spectroscopic (ICP-AES) elemental
used to minimize preferred orientation (15). (These effects            analyses conducted in a separate study (22). (In the latter,
were further decreased by using a fritted glass as the face            ICP-AES phase concentrations             were calculated from Ca,
plate.)                                                                Mg, and P concentrations in conjunction              with qualitative
   Peak position and intensity data were obtained by step              data from XRI) powder photographs. Colloidal apatite was
scanning specimens for predetermined 20 ranges (step size              assumed to be present in those samples where no phosphate
0.01#{176}28, s fixed time). Measured values (angle, count,
           10                                                          constituent     was detected      by XRD but where elemental
time) were recorded with a teletype (hard copy) and were               phosphorus      was determined with ICP-AES.)
simultaneously     stored for further processing with a Univac
1100/80 Series mainframe computer.                                     Discussion
   Single-phase      standard     samples     were continuously           A well-known limitation of qualitative XRD is its failure
scanned from 10 to 50#{176}20                and
                               at 2#{176}2a’mmn a chart recorder       to detect apatite at low concentrations,               particularly     in
speed of 2 cni/min. These traces were used for identification          infection-induced     stones. Strong struvite reflections overlap
purposes only and to ensure that the specimens remained in             almost all the identifiable apatite peaks. When corundum is
their original form. Five intensity values were determined             used as internal standard, the problem is aggravated                   by
for each reflection Ij, either by preparing new specimens or           overlap of the A1203 (012) peak with the apatite (002)
by scanning the same sample a second time. To check                    reflection. Only the very weak (100) and (111) apatite peaks
reproducibility of the sample preparation procedure, each              are then available for positive identification             of this com-
sample was mounted at least three times.                               pound. In such cases, the presence of apatite can be guessed
   A stepscan (0.02#{176}29, s) of the entire diffraction angle        only from a somewhat elevated background                  in the 30 to
range (10-50#{176}20) recorded twice for the same samples in                    region. This “amorphous
                                                                       34#{176}20                                halo” is often missed on
order to determine the relative intensities of all identifiable        powder photographs,        resulting in failure to detect apatite.
maxima. Peak positions and integrated             intensities were     The same difficulties exist in apatite/weddellite             mixtures.
established with the help of the program “xrowD”             (16, 17).                                                     has
                                                                       The tempering of apatitic calculi at 900 #{176}Cbeen suggest-
Peak intensities were determined from the chart recorder               ed to improve the crystallinity       of this compound and thereby
trace (0.5 cm/mm) and teletype printout.                               augment its diffracted intensity (23). However, the complex
   For the other preparations-i.e.,        mixtures of stone pow-      reactions and transformations         taking place at this tempera-
ders and corundum-about          five predetermined maxima per         ture preclude any other stone constituent from being simul-
phase were scanned. These reflections were later used in the           taneously identified. Another problem is that of identifying
quantitative analysis of calculi (Table 1).                            different kinds of apatites. Because this component always
   The intensities of these diffraction peaks were determined          occurs in a poorly crystallized state in biological concretions,
for all 50:50 (by wt) mixtures of the six stone components             the position of its diffraction maxima cannot be determined
and alpha-alumina.        We then processed data from these            with any accuracy. The differentiation between carbonate
samples with the “NBs*QuANr82” program package (18), to                 and hydroxyapatite      is thought to be of clinical significance
obtain the required reference intensity ratios.                         as the latter only forms in slightly acidic urine, while the
   After stone constituents were identified, those portions of         former precipitates      in a more alkaline medium. The dis-
                                                                       crimination     between the two apatite forms is thus impor-
                                                                       tant in the ascertainment         of an infected urine.
   Table 1. DiftractIon Maxima Used In the Analysis of
   Compound                                    hkl                                       Table 2. Reference Intensity Ratios (RIT)
Corundum                     012; 104; 110; 113; 024; 116                      Compound                          AlT          PublIshed value(s)
Whewellite                   lOT, 110; 020; 20; 112; 130, 41T
Weddellite                   200;211;  400; 222, 411; 510, 103; 213         Whewellite                          1.25
Hydroxyapatite               100; 111; 002; 211, 122, 300; 202;             Weddellite                          0.81
                               222; 213             -
                                                                            Hydroxyapatite                      0.45
                             020; 021; 111,041; 221; 131, 220 &             Brushite                            1.56
                               15T,20; 24T,022;151,24; 241                  Struvite                            0.90         1.0 (2t
Struvite                     110;011;111,021;130;012,211;                   Uric acid                           1.03         0.94 (21),       0b

                               040:112,022,211                                a Powder    Diffraction File card 31 -1982.             Filecard 21
                                                                                                                            Diffraction             -

Uric acid                    200;   210;   11;   400,   021,   T21          1959.

 290   CLINICAL CHEMISTRY, Vol. 34, No. 2, 1988
Table 3. Comparison of Constituent Concentrations (%                                  The quantitative     analysis for apatite, already hampered
  by wt) DerIved from QuantItative X-Ray Diffraction                               by the inadequaciesof its qualitative identification, is even
(Relative Intensity Ratio-Internal Standard Technique)                             more difficult. Homomorphous substitutions in the lattice of
                and Elemental Analyses                                             biological apatites-in     which someatomsare replaced with
                                                                                   other, similar atomsof different size-make        almost impossi-
  Stone                      Constituents                                          ble a meaningful       determination   ofconcentration.    Apart
sample no.                    IdentIfied             XRD             Elemental     from the resulting changes in lattice parameters,          which
                            Weddellite               49.3              50.7        cause some reflections to disappear and new ones to appear,
                            Apatite                   +                39.1        the absorption properties of the sample are changed. Artifi-
137                         Whewellite                8.1                9.6       cial apatite standards therefore seldom mimic the crystallo-
                            Uric acid                79.1                +         graphic properties of”real” biological apatites. This, togeth-
                                                                                   er with overlap problems in struvite and weddellite samples,
189                         Whewellite               24.9                +
                                                                                   is probably the main reason for the failure of XRD tech-
                            Weddellite               75.1                +
                                                                                   niques to give accurate values for apatite concentration.It is
207                         Whewellite               47.4                +         seen that apatite was not detected by XRD at low concentra-
                            Weddellite               52.6                +
                                                                                   tions (stones 265,402,415,426,458,461).        Indeed, XR.D data
                            Whewellite               13.3              22.4        failed to reveal apatite even in those stones where it had
237                         Apatite                  47.1              48.8        been detected in high concentrations        by ICP-AES (stones
                            Struvite                  8.2              25.0        268, 389). This discrepancy         between quantitative     XRD
264                         Apatite                  41.9              43.4        analysis   and ICP-AES data for apatite in stones 400 and 477
                            Struvite                 40.7              47.0        might be attributed     to differences in the microchemistry   of
                                                                                   hydroxyapatite    (standard) and the particular apatite in the
265                         Apatite                    -                11.1       iiohthS.
                            Struvite                 41.9               79.6
                                                                                       Reference intensity ratios were calculated     from peak
268                         Apatite                    +               46.2        heights (Table 2) and also from integrated   intensities. The
                            Struvite                 59.8              44.3
                                                                                   latter values varied, owing to the fact that in those cases
3fl                         Apatite                  53.3               54.3       where there was overlap of several peaks, the intensity of
                            Struvite                 20.1              31.7        the entiregroup of peaks was used in calculating           the
                            Whewellite                7.7                +         reference intensity       ratio constants. This entailed the re-
352                         Weddellite               47.6                +         scaling   of all relative   intensities depending on which maxi-
                            Uric acid                42.0                -         ma were included, which in turn affected the value of the
                            Whewellite               26.4               32.6       reference intensity ratio constant. However, analytical                re-
358                         Apatite                  46.2               47.5       sults are not affected by this approach as long as intensity
                            Struvite                 11.1               14.3       measurements are consistent within any one analytical
                                                                                   series. Nevertheless,       reference intensity ratios derived in
                            Whewellite               24.7                8.2
                                                                                   this way are of no use to other workers, because their values
389                         Weddellite               13.8                -
                            Apatite                    +                45.5       depend on arbitrary        selection of a number of peaks as the
                            Uric acid                23.7                -         “scaling cluster.” The rather close relationship between the
                                                                                   integrated    and peak intensities             observed in this study
10                          Apatite                  39.4               50.0       permit the reference intensity ratio constants listed in Table
                            Struvite                                    24.8
                                                                                   2 to be used with confidence by other stone analysts.                Peak
402                         Weddellite               92.1               85.1       intensities were also used by Otnes et al. (24) when they
                            Apatite                    -
                                                                                   applied the internal standard technique to determination                of
405                         Whewellite               43.2               44.9       stone components other than apatite. However, these work-
                            Uric acid                51.9                +         era did not publish their reference intensity ratio values and
                            Whewellite                 6.5               +         so we could not make a comparison                   with the constants
415                         Weddellite               62.9                +         obtained in the present study. Nevertheless,               comparison of
                            Apatite                    -                 8.5       data is essential, because the reference intensity ratios,
                                                                                   initially derived from chemically pure phases, have to be
                            Whewellite                 4.6               +
426                         Weddellite               95.4                +         adjusted as a result of the information                gained from the
                            Apatite                    -                 7.8       analysis of many calculi. The latter would take into account
                                                                                   the peculiar morphology of uroliths. Another way of deny-
                            Whewellite               41.0                +
                                                                                   ing better    reference      intensity      ratios would require the
458                         Weddellite               42.2                +
                            Apatite                    -                10.3       isolation of single constituents         from stones and their use in
                                                                                   the determination       of the constants.
                            Whewellite               29.9                +            The internal standard method yielded concentration                 val-
                            Weddellite               61.9                +
                                                                                   ues for the two preparations           of each specimen that agreed
                            Apatite                    -                17.1
                                                                                   within 10.6% (mean; range 1-21%). The mean agreement
                            Whewellite               30.4                +         obtained when averaging concentrations using one analyte
                            Weddellite               10.5                +         reflection and all standard            reflections was 8.5% (mean;
                            Apatite                  31.2                          range 0.7-17.1%). On the other hand, concentration                 agree-
                            Whewellite               21.9               37.3       ment ranged between 1.8 and 6.5% (mean 4.2%) when all
505                         Weddellite               28,3                -         analyte and standard         reflections were used. The benefit of
                            Uric acid                42.3                +
                                                                                   using multiple reflections and the resulting decrease in the
      a In %   (bywt); +, identified                   p
                                    butno quantification ossible; notidentified,
                                                                                   overall error is thereby illustrated.

                                                                                                      CLINICAL CHEMISTRY, Vol. 34, No. 2, 1988          291
    We did not establish detection limits in the present study,                             Table 4. Surveys of Urinary Calculus Analyses
owing to their extreme dependence on the matrix constitu-                                                                           Concn        %(bywt)
ents. Generally         it is thought that for a well-crystallized                   Survey       Sample       Constituents        expected        found
phase, concentrations           as low as 1% (by wt) may be deter-                   Si             A          Struvite               90             80
mined, but for poorly crystalline               phases, concentrations               1985                      Apatite                 10            20
>25% (by wt) may be completely undetected                   (25). Lagergren
(26) has reported that this applies to calcium oxalates where                                        B         Uric acid             100            100
1% (by wt) of whewellite              and weddellite can be distin-
                                                                                                     C         Xanthine              100            100
guished clearly. Schroeder and Bambauer (27) have report-
ed detection limits of the same order of magnitude (2-5%)                                            D         Whewellite             40             40
for brushite, octacalcium phosphate,              and whitlockite        in the                                Struvite               40             40
presence of other phosphates.            However, under unfavorable                                            Ammonium
conditions, such as line broadening,             these workers report a                                        acid urate             20             20
limit for apatite detection of 30-50% (by wt). Our results are                       S2              A         Struvite               90             80
in agreement with the latter study, in that no apatite could                         1985                      Apatite                10             20
be demonstrated           using XRD alone, but application of a
second technique revealed             its presence. Although apatite                                 B         Whewellite             60             60
                                                                                                               Struvite               20             20
could be identified from increased background readings in                                                      Apatite                20             20
very many cases, no quantitative analyses were possible.
Sutor and Scheidt (28) have published percentage detection                                           C         Whewellite             50             45
limits for stone constituents           in many two-component               mix-                               Uric acid              50             55
tures. Their results have shown that minor constituents                                              D         Xanthine              100            100
with concentrations in the range 5-20% (by wt) can remain
                                                                                     obtain convergence the computed constants.
                                                                                                             of                             This necessi-
undetected. Therefore, it can be deduced that when three-
                                                                                     tates longer measuring times, but position-sensitive          detec-
component mixtures are considered, even higher percent-
                                                                                     tors currently under development might prove helpful in
ages are likely to be missed.
                                                                                     this respect (37).
    Although the concept and underlying theory of quantita-
                                                                                        Thus, although results obtained           by quantitative    XE])
tive XE]) analysis are simple, practical details are more
                                                                                     analysis compare favorably with data obtained from other
complex. There are factors limiting the accuracy of the                              methods, we show here that XE]) analysis cannot be regard-
analyses as a result of several systematic                       errors (e.g.,
                                                                                     ed as a routine technique for the quantitative analysis of
preferred orientation, microabeorption),             random errors (par-             calculi. This is in agreement           with the findings of other
ticle statistics,     generator instabilities,       counting statistics),
                                                                                     researchers,     who have expressed the belief that a “conclu-
and calibration errors (improper matching of standard and
                                                                                     sion concerning the exact stone composition can only be
analyte in reference intensity ratio determinations                         with
                                                                                     reached after XE]) has been completed in conjunction with a
respect to impurities and crystallinity).
                                                                                     second procedure” (38). Indeed, when accurate elemental
    The present study revealed several factors that hamper
                                                                                     data obtained from techniques such as ICP-AES are used to
the proper implementation             of the reference intensity ratio-
                                                                                     facilitate   the quantitative     assessment   of phases identified
internal    standard      technique. Quantifying constants have to
                                                                                     by XRD, meaningful results can be obtained in conjunction
be determined by diffraction measurements                    of pure phases.         with even semiquantitative          XE]) analyses. The many ad-
In many cases these are not readily available, nor do they
                                                                                     vantages of such an approach have been discussed in detail
provide a close enough match to the analytes. This was
                                                                                     by Garbauskas        and Goehner (39). It is thus obvious “that
found to be one of the major shortcomings                  of the technique
                                                                                     the analyst’s time would be better spent on obtaining
as far as quantitative         apatite determinations         are concerned.
                                                                                     chemical information          than on making improvements in
A possible solution is to use calculated XRD data, where the
                                                                                     diffraction    data” (40). This view is supported by the results
necessary      constants     k1j are derived from structural parame-
                                                                                     obtained with such an approach through participation                in
ters only (29). This would enable the effect of element
                                                                                      two round-robin tests (urinary calculus analyses) organized
 substitutions     to be calculated, while all intensities would be
                                                                                      by the German Society for Clinical Chemistry (Table 4). It is
placed on an absolute scale (13, 30). However, a more
                                                                                      therefore concluded that semiquantitative           XE]) analysis,
 accurate knowledge            of the true spectral profile will be
                                                                                      supplemented by accurate quantitative          elemental data and
 required, and this is a goal that is still to be achieved.
                                                                                      a simple chemical (acid) test to distinguish hydroxy- and
 Furthermore,       while accurate integrated intensities are easi-
                                                                                      carbonate-apatite     (41), allows precise determination    of true
 ly obtainable from well-determined            structures,      peak intensi-
                                                                                      stone composition. For this purpose, easily counted peak
 ties are very difficult to synthesize (30,31). However, only
                                                                                      intensities, or even diffraction data recorded on a strip-chart
 peak intensities can be used in practice if analyses are to be
                                                                                      recorder, would suffice. Mixture series such as those report-
 promptly completed.
                                                                                      ed by Schneider et al. (4) would also be helpful in this
    Overlap of diffraction maxima is another factor that
 greatly impedes the possible application of quantitative
 XE]) to stone analysis. As with the calculation of theoretical                        The financial support of the Council for Scientific and Industrial
 powder patterns,          the use of whole-pattern             profile-fitting      Research (CSIR, South Africa), the Medical Research Council
 methods might be able to alleviate this problem (32, 33).                           (MRC, South Africa), and the University of Cape Town is gratefully
 When applying peak-separation              (34) and profile-fitting          (35,   acknowledged.
36) procedures         in the present study, we noted that very                      References
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