SOBECK & EBELING: MASS SPECTROMETRIC ANALYSIS FOR PHOSPHATE IN SOIL EXTRACTS 1

Mass Spectrometric Analysis for Phosphate in Soil Extracts; Comparison of

Mass Spectrometry, Colorimetry, and Inductively Coupled Plasma
Steven A. Sobeck* and Daniel D. Ebeling*

*Department of Chemistry, Wisconsin Lutheran College, Milwaukee, Wisconsin 53226

ABSTRACT

Phosphate (P) was extracted from soil samples using common soil extraction techniques: deionized water (DI)

and ammonium oxalate (AmOx). Colorimetry (Murphy Riley), electrospray ionization mass spectrometry

(ESI-MS), and inductively coupled plasma (ICP) were used in the P analysis. Mass spectrometry measured

considerably more phosphate than colorimetric analysis following DI extraction and showed a good

correlation with ICP for P analysis following AmOx extraction. These results suggest that ESI-MS analysis

may detect more than just inorganic P in soil extracts, as ionization and ion transport in ESI-MS may convert

organic P into detectable phosphate ions.

INTRODUCTION

Soil test P (STP) is used to determine the amount of phosphate (P) needed for crop production as well as to

determine environmental risks associated with elevated levels of soil P.1 Phosphorus is an environmental concern,

because excess P often promotes weed and algae growth in bodies of fresh water. Decomposition of the weed and

algae material reduces dissolved oxygen levels, leading to odors, fish kills, and a general degradation of the aesthetic

and recreational value of the environment.2,3,4

Nearly all methods of determining P in soil involve extraction of P into a liquid phase. Deionized water (DI) is

a commonly used extractant for P analysis,5 and ammonium oxalate (AmOx) is a multipurpose extractant that can be

used for cation analysis and P determination.6,7,8 Oxalate binds to cations, pulling some of them from soil particles.

An ammonium oxalate solution will extract more phosphate from soil than will DI, because the oxalate can replace

the phosphate at cationic sites on soil particles. AmOx extractions are of agronomic significance, because they more

closely approximate the P that is available to plants. DI extractable P is of particular environmental significance,

because it is an indicator of P run-off susceptibility.

 SOBECK & EBELING: MASS SPECTROMETRIC ANALYSIS FOR PHOSPHATE IN SOIL EXTRACTS 2

Inductively coupled plasma (ICP) optical emission spectroscopy and ascorbic acid colorimetry (Murphy Riley)

are commonly used techniques for P analysis in soil extracts. DI extracts are generally analyzed for P by

colorimetry or by ICP, and AmOx extracts are generally analyzed by ICP. In Murphy Riley colorimetric analysis,

phosphate binds to molybdenum to form a complex that has a blue color. The intensity of the blue color and the

absorption at 882 nm are proportional to the amount of phosphate available to form the complex. In ICP the sample

is atomized in a plasma for elemental analysis. Analysis by ICP typically results in slightly higher values for STP

than colorimetric methods,9 because some of the organically bound P can be unavailable to form the

phosphomolybdate complex necessary for color formation, whereas this ability is not an issue in ICP analysis as the

plasma releases elemental P in all P forms.

Mass spectrometric analysis of these types of soil extracts for P analysis is not a common technique. When it

has been used, it has generally taken the form of ICP-MS; that is, mass spectrometric determination of elements in

place of optical methods following ICP.10 Mass spectrometry has also been used for the analysis of speciation of

phosphate compounds after a separation technique.11 The way in which MS compares to other methods depends on

the amount of bound phosphates that are converted to inorganic phosphate during the electrospray process, because

the m/z of 97 is the only value that contributes to the phosphorus count. The mass spectrometric analysis described

in this manuscript is a simple electrospray ionization with a quadrupole detector used to analyze filtered extract for

inorganic phosphate as an alternative to the common colorimetric and ICP methods.

Three analysis methods and two extraction techniques are investigated in this manuscript. Two combinations

are not reported. The first omitted combination is colorimetric analysis of AmOx extracts, because in this lab work,

it was found that the blue color did not develop in attempts at colorimetric analysis of AmOx extractions. Searches

of scientific literature yielded no examples of colorimetric analysis of AmOx extracts for P. The AmOx extracts

were found to be analyzed by ICP, but when colorimetric analysis was employed alternate extractants were used.13

The other omitted combination is ICP analysis of DI extracts. The AmOx samples were sent out for ICP analysis as

a part of a different, funded research project. The DI extracts have not been afforded this opportunity. In this

research, DI extracts were analyzed by colorimetry and MS, and AmOx extracts were analyzed by ICP and MS.
 SOBECK & EBELING: MASS SPECTROMETRIC ANALYSIS FOR PHOSPHATE IN SOIL EXTRACTS 3

MATERIALS AND METHODS

Wisconsin soils tested. Selected soil samples from incubation studies by Ebeling et al. on Phosphorous Buffer

Capacity (PBC) of various Wisconsin soils13 (“PBC Inc” sample set) and the effect of biosolids application on soil

PBC14 (“BIO Inc” sample set) were used in this study.

Soil P extraction of Wisconsin soils. PBC Inc soil samples were extracted for soil P by a deionized water (DI)

extraction method5 with a soil:extractant ratio of 1:10. Samples were shaken in 50 mL conical tubes for 60 minutes

at 180 RPM on an orbital shaker. The tubes were centrifuged for 10 minutes at 2500 RPM. After centrifugation,

extracts were decanted and filtered. Filtered extracts were stored at room temperature for analysis by Murphy Riley

and MS.

BIO Inc soil samples were extracted for soil P by an ammonium oxalate (AmOx) extraction method5,15 with a

soil:extractant ratio of 1:20. Table 1 shows the components of the ammonium oxalate extractant. Samples were

shaken in 50 mL conical tubes for 2 hours in the dark at 180 RPM. The tubes were centrifuged for 20 minutes at

2000 RPM. After centrifugation, the extracts were decanted and filtered. Filtered extracts were stored in the dark at

3.0°C for analysis by ICP and MS.

Ascorbic acid colorimetric analysis using the Murphy Riley method. DI-extracted PBC Inc soils were

analyzed by ascorbic acid colorimetry (Murphy Riley).16 To prepare samples, 4.0 mL Reagent B (Table 1) and 19.0

mL DI water was added to 2.0 mL of each extract. Standards consisting of 5.0 mL of each standard P solution (0.1

ppm to 1.0 ppm P), 4.0 mL Reagent B, and 16.0 mL DI water and a 0.0 ppm P standard consisting of 4.0 mL

Reagent B and 21.0 mL DI water were also prepared. Samples were allowed 30 minutes for color development.

The absorbance of the samples and standard solutions at 882 nm was measured with a Perkin Elmer Lambda 25

UV/VIS spectrometer.

Table 1. Components of extractants and reagents used in AmOx soil extraction and Murphy Riley method.
Ammonium Oxalate Extractant Reagent A Reagent B
• 700 mL 0.2 M ammonium oxalate • 6 g ammonium molybdate • 1.584 g L-ascorbic acid
(56.8 g ammonium oxalte monohydrate in 2 L DI) • 0.146 g antimony potassium tartrate • 300 mL reagent A
• approx. 535 mL 0.2 M oxalic acid • 72 mL sulfuric acid
(36.0 g oxalic acid in 2 L DI) • brought to 1 L with DI water
 SOBECK & EBELING: MASS SPECTROMETRIC ANALYSIS FOR PHOSPHATE IN SOIL EXTRACTS 4

Table 2. Instrument specifications for the Varian 1200L Quadrupole MS for the electrospray ionization
mass spectrometry procedure. The instrument was run in negative mode using air as the nebulizing gas
and nitrogen as the drying gas.
Scan Mode Negative
Needle Voltage -4500 V
Electron Energy 70 eV
Detector Standard Gain 1270 V
Detector High Gain 1540 V
API Housing Temp 50°C
-6
Manifold 42°C, 3x10 Torr
Nebulizing Gas Air, 44 psi
Drying Gas N2, 16 psi, 200°C
Internal Air Temp 33°C

Inductively coupled plasma (ICP) optical emission spectroscopy. “Total elemental analysis” by ICP optical

emission spectroscopy on the AmOx-extracted BIO Inc soil samples was performed by the University of Wisconsin

Soil and Plant Analysis Lab (Madison, WI). The results from the P emission at 213.62 nm were used.

Electrospray ionization mass spectrometry (ESI-MS) procedure. A Varian 1200L Quadrupole MS

instrument was used to analyze samples. Table 2 shows instrument specifications. Samples were loaded into a 5.0

µL sample loop using the Varian 1200L-LC-ESI Switching Valve. A DI water mobile phase was set at a flow rate

of 0.1 mL/min to carry the sample to the electrospray. Varian MS Data Review software and Microsoft Office

Excel 2003 were used to analyze the raw data.

MS analysis of DI-extracted PBC Inc soil samples. Two sets of standard addition samples were prepared

from two DI-extracted PBC Inc soil samples: sample 6 (SA6) and sample 11 (SA11). Each standard addition set

consisted of six samples. Five samples consisted of 0.7 mL soil extract, 0.2 mL standard P solution (one in each

sample ranging from 3.4 to 31.6 ppm P), and 0.1 mL 350 ppm chlorate standard. The remaining sample consisted of

0.7 mL soil extract, 0.2 mL DI water, and 0.1 mL 350 ppm chlorate standard. These samples were analyzed by the

MS procedure described above using instrument specifications as listed in Table 2. The concentrations of P in SA6

and SA11 were determined from the standard addition peak areas.

DI-extracted PBC Inc samples were prepared using only a chlorate internal standard. Each sample consisted of

0.9 mL soil extract and 0.1 mL 350 ppm chlorate standard. These samples were analyzed by the MS procedure

described above using instrument specifications as listed in Table 2. The peak areas of these samples were then
 SOBECK & EBELING: MASS SPECTROMETRIC ANALYSIS FOR PHOSPHATE IN SOIL EXTRACTS 5

compared to the (peak area) to (P concentration) ratio of the reference sample (SA6 or SA11) in order to determine

concentrations of P.

MS analysis of AmOx-extracted BIO Inc soil samples. Two sets of standard addition samples were prepared

from two AmOx-extracted BIO Inc soil samples: sample 3 (SA3) and sample 9 (SA9). Each standard addition set

consisted of six samples. Five samples consisted of 0.8 mL soil extract and 0.2 mL standard P solution (one in each

sample ranging from 3.4 to 31.6 ppm P). The remaining sample consisted of 0.8 mL soil extract and 0.2 mL DI

water. No internal standard was added to the samples as the extractant itself (Ammonium Oxalate) functioned as an

internal standard. These samples were analyzed by the MS procedure described above using instrument

specifications as listed in Table 2. The concentrations of P in SA3 and SA9 were determined from the standard

addition peak areas.

AmOx-extracted BIO Inc samples were analyzed by the MS procedure described above using instrument

specifications as listed in Table 2 with no alterations made to the samples. The peak areas of these samples were

then compared to the (peak area) to (P concentration) ratio of the reference sample (SA3 or SA9) in order to

determine concentrations of P.

Murphy Riley and MS analysis of percent P in phytic acid. Phytic acid solutions ranging from 10 to 100

ppm phytic acid were prepared from a 50% weight-by-weight in water stock solution. Phytic acid solutions were

stored in the dark at 3.0°C prior to analysis. Phytic acid solutions were analyzed by Murphy Riley16 and MS using

the procedure described above and instrument specifications as listed in Table 2. Percent P was calculated by

dividing the number of phosphates detected by the theoretical number of phosphates that are bound to the phytic

acid rings.

Iron (Fe) precipitation of P in phosphate solutions. A control group and a test group were prepared for iron

precipitation. The control group consisted of four samples with 1.9 mL P solution (one in each ranging from 0.1

ppm to 5.0 ppm P) and 0.1 mL 350 ppm chlorate standard. The test group consisted of four samples with 1.7 mL P

solution (one in each ranging from 0.1 ppm to 5.0 ppm P), 0.1 mL 350 ppm chlorate standard, and 0.2 mL 500 ppm

Fe standard. These samples were analyzed by the MS procedure described above using instrument specifications as

listed in Table 2.
 SOBECK & EBELING: MASS SPECTROMETRIC ANALYSIS FOR PHOSPHATE IN SOIL EXTRACTS 6

Standards. Phosphate standards (standard P solutions) ranging from 3.4 ppm P to 31.6 ppm P were prepared

by serial dilution in DI water starting from a concentrated phosphoric acid solution. All phosphate solutions were

then standardized by Murphy Riley.16

A 350 ppm chlorate standard was prepared by dissolving 0.2567 g potassium chlorate in 500 mL DI water.

This solution was used as an internal standard to correct for variability in the Varian MS instrument and to

normalize ion peaks in the MS spectrum.

RESULTS AND DISCUSSION

Iron precipitation of phosphate in phosphate solutions. Iron precipitation of P in soil extracts was

performed in order to determine if the MS peak at 97 m/z was due to phosphate and not another ion. Results showed

that after addition of iron, the ion peak at 97 m/z was eliminated or significantly reduced. This indicates that MS

was likely detecting phosphate.

Matrix effects and the use of standard addition. An attempt was made to quantify P in the soil extracts by

establishing a standard curve in MS using P solutions in DI water. Peak areas for soil samples analyzed by MS

would have been compared to the standard curve to determine P concentration, if there were no complicating matrix

effects. Differences in matrix effects can be gauged by comparison of the slopes of the standard addition curves to

the P standard curve. The slope of the P standard curve was approximately 5 times greater than the slope of the DI-

extracted PBC Inc standard addition curve and 50 times greater than the slope of the AmOx-extracted BIO Inc

standard addition curve. This indicates a significant matrix effect, likely due to components extracted from the soil

and the ammonium and oxalate ions. Electrospray performance is very dependent on the amount and type of salts

that may be in the matrix. Due to these matrix effects, two samples for each extractant were chosen for standard

addition analysis. In order to minimize analysis time, the remainder of the samples were analyzed without standard

addition and compared to the reference (standard addition) samples (as described above). For this to be valid, the

matrix effects cannot significantly differ between soil extract samples. The reference samples in the DI-extracted

PBC Inc sample set had slopes of 0.0038 and 0.0045 (normalized peak area/ppm P) for SA6 and SA11, respectively.

For the AmOx-extracted BIO Inc sample set, the slopes of the reference samples were 0.0035 and 0.0051

(normalized peak area/ppm P) for SA3 and SA9, respectively. This demonstrates a difference in the intensity of

signal response to phosphate due to matrix effects. The resultant discrepancy between P values based on the
 SOBECK & EBELING: MASS SPECTROMETRIC ANALYSIS FOR PHOSPHATE IN SOIL EXTRACTS 7

different reference samples (Fig. 1 and 2) clearly indicates that in the future, the matrix effects must be controlled if

one does not wish to run standard additions on every sample.

Comparison of soil P in DI-extracted PBC Inc soils measured by Murphy Riley and MS. Mass

spectrometry measured considerably higher concentrations of P than Murphy Riley in the DI-extracted PBC Inc soil

samples (Fig. 1). On average, MS measured 4200% higher when using SA6 as a reference sample and 3200%

higher when using SA11. The correlation between Murphy Riley and both MS data sets was poor (R2 = 0.1281).

This may be because MS detects more P that is unavailable for phosphomolybdate complex formation.

18

16

14

12
Murphy Riley
ppm P

10
MS - SA6
8 MS - SA11

0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Sample #

Fig. 1. Comparison of soil P in DI-extracted PBC Inc soils measured by Murphy Riley and MS. SA6 and
SA11 are used as reference samples for MS in order to calculate the concentrations in samples 1-15. MS
error bars represent the standard deviations for n=3.

Comparison of soil P in AmOx-extracted BIO Inc soils measured by ICP and MS. Mass spectrometry

measured similar concentrations of P as ICP in the AmOx-extracted BIO Inc soil samples when using SA9 as a

reference sample (Fig. 2). However, when using SA3 as a reference sample, concentrations of P measured by MS

were 270% higher than the “MS - SA9” data set. The correlation of MS to ICP data was R2 = 0.8827. The change in

the absolute concentrations is likely due to matrix effects as mentioned above. The relatively good correlation

between ICP and MS in AmOx may result from a similarity in the amount and types of phosphorus species that the

techniques can detect.

 SOBECK & EBELING: MASS SPECTROMETRIC ANALYSIS FOR PHOSPHATE IN SOIL EXTRACTS 8

120.0

100.0

80.0
ICP
ppm P

60.0 MS - SA3
MS - SA9

40.0

20.0

0.0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Sample #

Fig. 2 Comparison of soil P in AmOx-extracted BIO Inc soils measured by ICP and MS. SA9 and SA 3 are
used as reference samples for MS in order to calculate the concentrations in samples 1-15. MS error bars
represent the standard deviations for n=3.

Comparison of percent P in phytic acid measured by Murphy Riley and MS. Phytic acid is a six-carbon

ring with a phosphate bound to each carbon. It serves as a good example of organic phosphate.

Mass spectrometry measured a higher percentage of phosphate in phytic acid than Murphy Riley. On average,

MS measured 23% P in phytic acid, whereas Murphy Riley only measured 13%. These results are expected as

electrospray ionization (ESI) is a much “harder” technique than Murphy Riley and is thus liable to break apart larger

molecules. Although ESI is often considered a “soft” MS technique, the analyte is propelled quickly through a part

of the spectrometer that is held at atmospheric pressure. The collisions of phytic acid ions with the gas molecules

likely remove phosphate ions from the carbon ring. These phosphate ions are thus detected as inorganic phosphate.

It is expected that the carbon ring will be stripped of some phosphate ions during the molybdate reduction

reaction in the Murphy Riley method; however, this should not produce as much inorganic P as in the MS

procedure, as observed. Also, natural degradation of phytic acid to produce inorganic P is expected, even in the

reagent bottle. Thus the phytic acid solution may consist of a certain percent inorganic P prior to analysis. The

percent P measured by Murphy Riley may be representative of this “natural” degradation percentage.

These results support the hypothesis that mass spectrometry measures P not detected by the Murphy Riley.
 SOBECK & EBELING: MASS SPECTROMETRIC ANALYSIS FOR PHOSPHATE IN SOIL EXTRACTS 9

CONCLUSION

Electrospray ionization mass spectrometry has promise as a technique for the analysis of phosphate in soils. The

problem of varying matrix effects may be a limitation in the applicability of MS analysis for P. Future work should

be invested into improving the precision of MS phosphate measurements and minimizing matrix effect variation;

also, measuring P for the same soils after different extraction techniques would give a useful comparison.

The value of STP in soil extracts varies with measurement technique. Just as extraction methods operationally

define STP, the method of analysis of P in those extracts has a similar impact. It appears that MS converts organic P

into inorganic P and detects more phosphate than Murphy Riley analysis. With further improvements to the method,

MS may be found to play an important role in providing a unique and important value of STP, such as

approximating bioavailable or run-off susceptible P.

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