ANALYTIOAL BIOCHEMISTRl’ !

ht, 443447 (1968)

A Method for Quantitative Determination of Phosphonate

Phosphorus in the Presence of Organic and
Inorganic Phosphates1
J. A. AALBERS” AND L. L. BIEBER
Department of Biochemistry, Michigan State University,
East Lansing, Michigan 48823

Received October 16, 1967

Several laboratories have reported the natural occurrence of com-

pounds containing a carbon-phosphorus bond. Such bonds are stable to
prolonged treatment with hydrochloric acid. This stability can be used as
an aid in the identification of phosphonates. For example, Horiguchi and
Kandatsu (1) proved the natural occurrence of aminoethylphosphonic
acid by treating samples for several hours with hydrochloric acid to
hydrolyze phosphate esters. The unhydrolyzed phosphonates were then
separated from phosphate by chromatography. The amount of phosphate
or nitrogen in the fractions isolated by column chromatography has been
used for quantitative determination of phosphonates (2, 3). Such
analytical methods are accurate but required considerable time for each
analysis.
In our studies on the incorporation of DMAP3 and TMAP into Musca
domestica larvae lipids (4), a method for the rapid estimation of small
amounts of phosphonolipid phosphorus in the presence of lipids con-
taining phosphodiester and phosphomonoester bonds was desirable. In
initial experiments it was observed that sulfuric acid/H202 digestion, as
described by Bartlett (5)) resulted in limited cleavage of the carbon-phos-
phorus bond of phosphonolipids. If a method for total phosphorus, in-
cluding phosphonate phosphorus, was used together with a method that
did not hydrolyze the carbon-phosphorus bond, then phosphonate phos-
3Research paper No. 4182 from the Michigan State Agricultural Experiment
Station. This research was supported by Grant 5 ROl AM16514 from the Divi-
sion of Arthritis and Metabolic Diseases,United States Public Health Service.
a NSF undergraduate fellow.
* Abbreviations used: DMAP, dimethylaminoethylphosphonic acid; TMAP, tri-
methylaminoethylphosphonic acid ; APP, 3-aminopropylphosphonic acid; AEP, 2-
aminoethylphosphonic acid.
443
444 AALBERS AND BIEBER

phorus could be determined by difference. In this paper, such a method

is described.

MATERIALS AND METHODS

DMAP was the generous gift of Dr. A. F. Isbell, Department of
Chemistry, Texas A and M University, and TMAP was kindly provided
by Dr. A. F. Rosenthal, Long Island Jewish Hospital. APP and AEP
were purchased from California Biochemical Corporation.
The amount of phosphorus present in phosphodiester and phosphomono-
ester linkage was determined by treating the samples with sulfuric acid
as decribed by Bartlett (5) : 0.5 ml of 10 N H&SO, was added to the dried
lipid samples, followed by heating in an oven at 160’ for 3 hr. After
cooling, 0.1 ml H,Oz was added, and the samples were heated at 160’
for 1.5 hr. The total phosphate in the samples, including phosphonates,
was determined by hydrolyzing the dried sample in 0.4 ml of 70% HClO,
at 17.5’ for 16 hr. After cooling, 0.3 ml of 10 N HzS04 and two drops of
30% Hz02 were added and the samples heated at 175’ for 2 hr. Ammo-
nium molybdate was added, and the color developed as described by
Bartlett (5). Because of the additional hydrogen ion from perchloric acid,
0.3 ml of 10 N HzSO, was used rather than 0.5 ml as described above for
the H,SO, digestion. When 0.5 ml of 10 N H,SO, was used, difhculty was
encountered with the HCIO, method, presumably due to excess acid. Ash-
ing the phosphonates with Mg (NOa) 2, as described by Ames (6)) was also
tested. After ashing, 0.5 ml of 10 N H,SOa was added, and the color devel-
oped as described by Bartlett (5). The ashing technique is faster and data
comparable to those of the HCIO, assay can be obtained. The combina-
tion of HCIOI and HzOz is potentially explosive; therefore, heating was
performed in an oven located in a closed hood.
KH,PO, was used as the standard. A standard phosphate curve was
included with every determination. The optical density at 830 q was
determined with a Hitachi spectrophotometer using a 10 mm light path.

RESULTS AND DISCUSSION

As shown in Figures 1 and 2, the OD at 830 rn,u. is linear from 0.01 to

0.1 eole of phosphate when either digestion with HClO, or ashing with
Mg(NO,)z is used. The amount of phosphate is identical to that which
can be determined by the method of Bartlett (5). If large amounts of
organic materials are present and the Mg(NOs) 2 method is used, the
samples should be ashed twice.
Digestion with either HClO, or Mg(N0,) 2 can be used for determining
the amount of phosphorus present in water-insoluble materials such as
 QUANTITATIVE DETElBMINATION OF PHOSPHONATFiS 446

I I I I
- .=AEP
o,40- m = APP

E - l =DMAP
o,40- 8 = TMAP

0.02 0.06
flmoles P
Fra. 1. Linearity of HClO, assay with phosphonates. Dried samples digested
with HClOd ss described under “Methods.”

phosphonolipids. The difference between the amount of phosphate ob-

tained by the HCIO, assay and the HzS04 assay has been used in this
laboratory for quantitative estimation of the amount of TMAP and
DMAP present in housefly lipids (4)) i.e., &moles PO, (HCIOa or

- .=AEP
o.40- m =APP

0.20 -

i3 - n = TMAP
l = DMAP
0.40-

0.20-

002 0.06 0.10

umoles P
FX 2. Linearity of Mg(NO& assay with phosphonates. Dried samplea were
ashed with Mg(NO.1, and the color developed aa described under “Methods.”
446 AALBEFtS AND BIEBFB

TABLE 1
Limited Hydrolysis of Phosphonntes by Controlled H2SOd Digestion
pm&s
Compound added H:O¶ added

DMAP - 0.25 0.0625 1

+ 0.25 0.6675 3
TMAP - 0.175 0.075 4
+ 0.175 0.075 4
AHP - 0.175 0.0925 1
+ 0.175 0.010 6
APP - 0.25 0.998 2
+ 0.25 0.064 16
Phosphorylcholme - 0.091 0.090 99
+ 0.091 0.093 102

The samples were digested at 16O”C, as described under “Methods.” Where indicated,
H202 was omitted; otherwise, the samples were treated identicahy to those with HzO~.
The values given in the table represent an average of at least 10 determinations.
Mg(N0,) 2 assay) minus @moles PO, (H&SO4 assay) = qoles P con-
taining a carbon-phosphorus bond.
When very accurate data are needed, a correction for the limited
cleavage of the carbon-phosphorus bonds by HzS04 (< 5% for DMAP
and TMAP) can be made by determining the per cent hydrolysis of
known amounts of phosphonate. As shown in Table 1, the phosphonates
vary in stability to H,SO,, especially when H,Oz is added. Approximately
16% conversion of APP to inorganic phosphate was found when H,Oz was
added, but only 2% in the absence of HzOz. Thus, some carbon-phosphorus
bonds are not stable to the H2S04 treatment. As shown in Table 2, the
phosphorus present in dil%.rltly hydrolyzable esters such as 3-phospho-
glyceric acid is quantitatively converted to inorganic phosphate. The
results are consistent with the premise that the procedures used convert
phosphate esters to inorganic phosphate.
The total digestion time in HClO, reported here is greater than that
used by other investigators. Frequently, digestions with HCIOa such as
TAl3LE 2
Conversion of Difficultly Hydrolyzable Phosphate Esters to
Pi by the HC104, the Mg(NOa)t, and the HzSO4 Methods
pm&s Pi detected
&moles
Phosphate ester tegted added HClO, assay HaSO4 assay MgNOs assay

3-Phosphoglyceric acid 0.10 0.10 0.10 0.10

a-Glycerophosphate 0.10 0.11 0.10 0.09
0-Phosphoethanolamine 0.10 0.10 0.10 0.10

Phosphate analyses were done as described under “Methods.”

 QUANTITATIW DETEEUINATION OF PHOSPHONATES 441

in the method described by Galanos and Kapoulas (7) require heating

individual samples over an open flame. In our investigations, the
simultaneous assay of many fractions obtained from silicic acid chro-
matography of larval phospholipids was required so a method that did
not utilize individual heating of samples was desirabble. No effort was
made to determine the minimum time required to hydrolyze the phos-
phonates at 175” in HClO,.
SUMMARY
A method for quantitative estimation of the phosphate present in com-
pounds containing a carbon-phosphorus bond is described, Two phos-
phorus assays are employed. One assay is for total phosphate, which
can be determined by digesting with perchloric acid or ashing with
Mg(NO,),. The other assay is for total nonphosphonate phosphorus,
which can be determined by controlled digestion in H,SO,. The difference
between total phosphate and the phosphate determined by controlled
HzSO, hydrolysis represents the amount of phosphorus present in a ear-
bon-phosphorus linkage.
REFERENCES
1. HOIUGUCHI, M., AND KANDATSU, M., Nature 184, 991 (1956).
2. LIANQ, C. R., AND ROSENBERG, H., Biocbn. Biophys. Acta 125, 548 (1966).
3. KITTREDGE, J. S., ISBELL, A. F., AND HUGHES, R. R., Biochemistry 6, 269 (1967).
4. BIEBER, L. L., Biochim. Biophys. Acta (in press).
5. BARTLETT, G. R., J. Viol. Chem. 234, 466 (1959).
6. AMES, B. N., in “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan
eds.), Vol. VIII, p. 115. Academic Press, New York, 1966.
7. GALANOS, D. S., AND K~POULAS, V. M., Anal. Chim. Acta 34, 360 (1966).