CLIN. CHEM.

23/12, 2288-2291 (1977)

4-Nitrophenolin 4-NitrophenylPhosphate,a Substratefor Alkaline

Phosphatase,as Measuredby Paired-IonHigh-PerformanceLiquid
Chromatography
Paula H. Culbreth, Irma W. Duncan, and Carl A. Burtis

We used paired-ion high-performance

liquid chromatography to determine the 4-nitrophenol content of 4-nitrophenyl phosphate, a substrate for alkaline phosphatase
analysis. This was done on a reversed-phase column with
a mobile phase of methanol/water,
45/55 by vol, containing 3 ml of tetrabutylammonium phosphate reagent per
200 ml of solvent. At a flow rate of 1 mI/mm, 4-nitrophenol
was eluted at 9 mm and monitored at 404 nm; 4-nitrophenyl
phosphate was eluted at 5 mm and could be monitored at
311 nm. Samples of 4-nitrophenyl phosphate obtained
from several sources contained 0.3 to 7.8 mole of 4nitrophenol per mole of 4-nitrophenyl phosphate.
AdditIonal Keyphrases:
enzyme activity

analytical error
reagent purity

quality con trol

phosphate)

to the nonpolar column packing, thus selectively retaining ionic species on the column (3). The pH of the
buffered mobile phase, 7.5-8.0, maintains the counter-ion and the sample in an ionic form.

Materials and Methods1

Reagents
Disodium
4-nitrophenyl
phosphate
hexahydrate
(4NPP) was purchased
from five sources: Aldrich
Chemical Co., Inc., Milwaukee,
Wis. 53233; Biodynamics/bmc,

Paired-ion high-performance
liquid chromatography
has been used to determine the 4-nitrophenol
(4NP)
content of 4-nitrophenyl phosphate (4NPP), a substrate
used for the assay of alkaline phosphatase. In the assay
for this enzyme, the absorbance of 4NP in alkaline solution, produced by the hydrolysis of 4NPP, is measured
at 404 nm. The 4NP as an impurity may interfere with
the enzymatic analysis and also lead to a high background absorbance
during the spectrophotometric
measurement.
According to a Selected Method for alkaline phosphatase,
the purity of 4NPP with respect to
4NP is determined by measuring the absorbance of the
substrate at 415 nm under certain conditions of concentration,
pH, and temperature
(1, 2). If the absorbance at 415 nm is below a specified limit, the 4NPP, with
respect to 4NP content, is considered to be suitable for
use as the substrate for alkaline phosphatase.
Here, we describe a more specific method for determining the 4NP content of 4NPP.
The 4NP and 4NPP are separated on a reversedphase column by paired-ion chromatography
in which
a large organic counter-ion
(tetrabutylammonium

added to the mobile phase forms an ionic

complex with the compound

to be analyzed.
Presumably, the alkyl portion of the ionic complex is attracted

Indianapolis,

Ind.

46250;

ICN

Pharma-

ceuticals, Inc., Cleveland, Ohio 44128; Sigma Chemical

Co., St. Louis, Mo. 63178 (two lots); and Technicon
Instruments
Corp., Tarrytown, N. Y. 10591 (two lots).
The 4-nitrophenol
(4NP) was purchased from Calbiochem, La Jolla, Calif. 92037. 2,4-Dinitrophenol,
2,5dinitrophenol,
and 2,6-dinitrophenol
were purchased
from Aldrich Chemical Co., Milwaukee, Wis. 53233.
2,4,6-Trinitrophenol
was purchased from J.T. Baker
Chemical Co., Phillipsburg,
N. J. 08865. Spectrophotometric grade methanol was obtained from Burdick
and Jackson, Muskegon, Mich. 49442. Water was deionized and glass distilled before use.
Chromatographic

System

The system used for the chromatographic

analyses
included a Model 4200 liquid chromatograph
(Varian
Associates,
Palo Alto, Calif. 94303) equipped with a
Model U6K injector (Waters Associates, Milford, Mass.
01757); a Waters 1sBondapak C18 reversed-phase
column (30 cm X 4 mm) preceded by a precolumn (5 cm X
2 mm packed with Waters Cis/Porasil
B, 37-75 urn,
used to prolong useful life of column); a Varian VanChrom variable wavelength detector; and a Varian A-25

Bureau of
Service,

Laboratories,
Center for Disease Control, Public Health
U. S. Dept. of Health, Education, and Welfare, Atlanta, Ga.

30333.
Received June 10, 1977; accepted

2288

Sept. 21, 1977.

CLINICALCHEMISTRY,Vol. 23, No. 12, 1977

Use of trade names is for identification

stitute

endorsement

by the USPHS

only and does not conor by the U. S. DHEW.

B
4NPP

(31mm)1

4NP

4NP

4NP injected, nmol

mmol 4NP/mole 4NPP8
CV, %

0246810

(A) 311 nm and 0.1 A full scale; (B) 404 nm and 0.5 A full scale. Other variables:
mobile phase, methanol/water, 45/55 by vol. pIus 3 ml of tetrabutylammonium
phosphate reagent per 200 ml of solvent; 1mI/mm flow rate; Bondapak C,8
reversed-phase column; 20-al injections. AU, absorbance unit

0
0

4
4
w
0.

0.27
1.00

2.43
9.00

2.8

2.0

270 nmol (100 gig) of 4NPP. The 4NP was quantitated

by comparing the peak area for the 4NP eluted from a
sample with that from 30 ui of a standard
solution
containing 1.96 nmol (272 ng) of 4NP. Standards were
analyzed immediately
before the manufacturers
samples. The results were expressed as mmol of 4 NP/mole
of 4NPP.
To demonstrate
the resolution
obtained
by the
chromatographic
analysis, we analyzed 36 u1 of a synthetic
mixture
containing
4NPP (2 nmoi), 4NP (4
nmol), 2,6-dinitrophenol
(6 nmoi), 2,4-dinitrophenol
(4
nmol), 2,5-dinitrophenol
(10 nmol), 2,4,6-trinitrophenol
(6 nmol), and bis(4-nitrophenyl)
phosphate (2 nmol),
and monitored the column eluent at 311 nm for 55 mm.
Two microliters of phenol (2 nmol) was analyzed separately and detected at 218 nm.
Spectrophotometric

300

400

2.16

2.8

100

4NP INJECTED(ng)
007
0.6
018

0.14
0.5
4.6

-4---

Fig. 1. Chromatograms for 4NPP (1.75 nmol, peak at 5 mm) and

4NP (10.43 nmol, peak at 9 mm)

1025 50

0.08
0.3
5.2

0Millimolesof 4NP injected/270 X 10#{176}.

This is calculated as if the 4NP were
present In 270 nmol of ANPP and isa model for the calculations performed for
Table 2.

(404nm)

L.
0246810
TIME (mm)

Table 1. ReproducibIlity of Chromatographic

Determinations of 4-Nitrophenol Standards for
Five Successive Injections

0.72
4NP INJECTED(nmol)

Fig. 2. Linearity of detector response for 4NP at 404 nm and 0.1

A full scale
equation for the line Is y = 7.9685 (SD, 0.07)x - 0.4756 (SD, 0.11),
from duplicate analyses of seven concentrations, with 30-zl injections

The

recorder. A Varian CDS 101 integrator

was used to
record the peak area electronically
from the detector
signal.
The mobile phase used for the chrornatographic
separation was methanol/water,
45/55 by vol, plus 3 ml
of tetrabutylammonium
phosphate (PlC Reagent A
from Waters Associates) per 200 ml of solvent. The
mobile phase contains about 5 mmol of tetrabutylammonium phosphate per liter, and has a pH of 7.5-8.0.
The solvent was filtered through a membrane of average
pore size 0.45 urn to degas it and remove small particles.
Procedure
At a flow rate of 1 rnl/min with a pressure of 10.3517.25 MPa (1500-2500 psi), the column eluent was
monitored at 404 nm for 4NP. Samples of 4NPP from
the various sources were analyzed for 4NP by injecting
30 ul of an aqueous solution of each sample containing

Measurement

of 4NP

To compare the performance of the chromatographic

method with that of an accepted procedure, we assayed
a series of 4NPP samples that had been supplemented
with known quantities of 4NP, by both the chromatographic method and a spectrophotometric
method as
described by Bowers and McComb (1), based on the
spectral properties
of 4NP at 415 nm. Both a Cary
Model 118 Spectrophotometer
(Varian Associates) and
a Model MS2 Micromedic
Spectrophotometer
(Micromedic Systems, Inc., Horsham, Pa. 19044) were used
for the spectrophotornetric
measurement.
The 4NPP
samples were prepared to cover a concentration
range
of 4NP from 0.25 to 8.00 mmol of 4NP/mole of 4NPP.
In performing the spectrophotometric
measurements
at 415 nm we found it necessary to dilute the 4NP
samples containing more than 1.00 mrnol of 4NP/mole
of 4NPP. Chromatographic
analyses were performed
by injecting 30 ui of a 12.5-fold dilution of each of the
prepared

samples.

Results and Discussion

Figure 1A illustrates a typical separation
obtained
from the chromatographic
analysis of a mixture of
4NPP and 4NP by monitoring the column eluent at 311
nm (the Xmax for 4NPP). By the comparison of analyses
of duplicate
samples
containing
4NPP and 4NP, a
fivefold increase in sensitivity for 4NP is observed at 404
nm (Figure 1B) over that obtained at 311 nm (Figure
1A). At 404 nm, the sensitivity for 4NPP is so decreased
that a peak corresponding
to 10 nmol is not visible in
CLINICALCHEMISTRY,Vol. 23. No. 12, 1977

2289

Table 2. 4-Nitrophenol Content of 4-Nitropheny I Phosphate from Various Sources

Sample
Source
4NP, mmol/mole
of 4NPP

Technicon8

B
ICN

Aldrich

BMC

Sigma8

Sigmab

Techniconb

776d

1.00

0.53

0.27

<0.26

<0.26

<0.26

alot 1. 5Lot 2. #{176}This

sample was more than a year old.

Table 3. Comparison of High-Performance Liquid

Chromatographic and Spectrophotometric
Determinations of 4-Nitrophenol in 4-Nitrophenyl
Phosphate

T001
11404nm

II

Mililmol 4NP
added per mole
of 4NPP

4NP

MillImoles of
Chromatography

0
0.25

0J__

0.26

0.50
0246810

0246810

0246810

1.00
2.00

0246810

TIME mn)

0.44
1.00

0.50

0.47

0.99

1.98

2.00

0.94
1.94

3.96

4.00

3.88

7.92

7.90

7.44

4.00
8.00

Below limit of detection.

a
0246810

0246810

8 10

8 10

Fig.3. Chromatograms for 4NPP from different sources at 404

nm and 0.1 A full scale; 30-uI injections
variables are the same as in Fig. 1. The 4NP standard (Sm) represents
1.96 nmol (272 ng); A-(3 show various amounts of 4NP In samples of 270 nmol
(100 g) of 4NPP. AU, absorbance unit
Other

Other values obtained with the Caiy 118 are corrected by this amount of 4NP
found ;n the 4NPP used.
#{176}Other
values obtained with the Micromedic MS2 are corrected by this amount

of 4NP

Figure 1B, although a peak is evident (as seen in later

figures) for larger amounts of 4NPP. For detection of
trace amounts of 4NP, the manufacturers
samples were
analyzed at 404 nm. Linearity of detector response for
4NP at 404 nm is demonstrated
in Figure 2. At an absorbance range of 0.1 A full scale, the lower limit of detection of 4NP is 0.07 nmol (10 ng) with a signal-to-noise
ratio of 5/1 and the reproducibility
for five successive
injections
is shown

of 4NP standards
in Table 1.

in various

The nonenzymatic

hydrolysis

of the 4NPP

and the

2 Other criteria for the purity of 4NPP that must also be considered
are: enzyme activity, mole fraction of inorganic phosphate contamination, and pH of a 60 nmol/Iiter aqueous solution (1, 2).

2290

CLINICALCHEMISTRY, Vol. 23, No. 12, 1977

found in the 4NPP used.

Table 4. Retention Times of 4-Nitrophenyl

Phosphate, 4-Nitrophenol and Related
Compounds a
Solvent

Sample
peak

RetentIon time, mln

4-Nitrophenyl phosphate

concentrations

Figure 3 shows chrornatograms

of seven lots of 4NPP
obtained from the five different sources. The free 4NP
content of 4NPP corresponding to the samples in Figure
3 is shown in Table 2. The limit of acceptability
is considered to be 1.00 rnrnol of 4NP per mole of 4NPP (1,
2). 2 Sample A, containing
the largest amount of 4NP,
was more than a year old. Sample G, newly acquired
from the same source as sample A, contained
very little
4NP, suggesting
that sample A had undergone
significant hydrolysis
with time. All other samples were below
the limit of acceptability of 1.00 mmol of 4NP per mole
of 4NPP. The double peak (Figure 3), caused by the
large amount of 4NPP injected to detect a trace amount
of 4NP, does not interfere with the quantitation
of
4NP.

mole of 4NPP
Spectrophotometry
Cary 118
Mlcromedlc MS2
0.22
0.20
0.30
0.23

4NP found per

5.4
6.7

Phenol
4-Nitrophenol
2,6-Dinitrophenol

9.1
10.8

2,4-Dinitrophenol

13.2

2,5-Dinitrophenol
2,4,6-Trinitrophenol

14.6
28.4
51.0

Bis(4-Nitrophenyl)
8

Run conditions

phosphate

are the same as in Fig. 1. with detection at 311 nm and 36

M1injected.

Analyzed

absorbance
hydrolysis

separately

at 218 nm.

of 4NP
of 4NPP

are both sensitive

to pH (4). No
was detected
during the analysis.

The analysis of 4NP standards

immediately

before the

analysis of manufacturers
samples of 4NPP and with
the same mobile phase assures comparison
at the same

pH.
The results

of the determination

of 4NP in 4NPP

by

chromatography
and by the spectrophotometric
method
of Bowers and McComb, with use of two different
spectrophotometers,
are given in Table 3. Similar results
were obtained with both methods. It should be noted
that
that

4NPP samples
may contain other contaminants
also absorb at 415 nm. Such contaminants
would

result in erroneously high values for 4NP when measured by the spectrophotometric
method. In addition,
other contaminants
such as bis(4-nitrophenyl)
phosphate are found in 4NPP samples, which do not absorb
at 415 nm but which inhibit enzyme activity in alkaline
phosphatase
analysis.3 With 4NPP samples that contain such contaminants,
the chromatographic
method
would be the preferred method because it would provide
for separation, detection, and quantitation
of most organic contaminants
in a 4NPP sample. For example, as
shown in Table 4, several compounds related to nitrophenol can be separated from 4NPP under the conditions described. In these separations the column eluent
was monitored at 311 nm to detect the 4NPP, the bis(4nitrophenyl)
phosphate,4
and the other compounds
listed with the exception of the phenol, which was detected at 218 nm. The other compounds related to nitrophenol could also be detected at 404 nm. Under the
chromatographic
conditions utilized, the bis compound
was eluted in 51 mm. However, this elution time can be

shortened
significantly
by increasing
the methanol
concentration
in the mobile phase.
For the detection of 4NP in 4NPP, the chromatographic procedure described here is more.specific than
is the spectrophotometric
method. The clinical chemist
can use this method to investigate the 4NP content of
new lots of 4NPP as well as to monitor one particular
lot that is being used over a long time. In addition, the
manufacturer
may use it for quality control in the production of 4NPP.
We gratefully
Sampson

acknowledge

the technical

assistance

of Dr. Eric J.

G. N., Jr., and McComb, R. B.,

Measurement

of total a!1988

and Louis E. Seibert.

References
1. Bowers,

kaline

phosphatase

activity

in human

serum. Clin. Chem. 21,

(1975).
2. Bowers, G. N., Jr., McComb,
R. B., and Horder, M., Reagent
quality, a prerequisite for meaningful measurements
in clinical enzymology. In Proc. 3rd. mt. Cong. Prospective Bid., Pont-#{225}-Mousson,

France, 1975, p 257.

3. Paired-ion chromatography:
with Drs. George Bowers and Robert
McComb concerning bis(4-nitrophenyl) phosphate.
4We determined a X,,, for the bis(4-nitrophenyl)
phosphate to be
at 287 nm. However, it can be detected at 311 nm.
Oral

communication

An alternative to ion-exchange.
Waters Associates, Milford, Mass., publication no. D61, May 1976.
4. Kirby, A. J., and Jencks, W. P., The reactivity of nucleophilic reagents toward the p-nitrophenyl phosphate dianion. J. Am. Chem.
Soc. 87, 3209 (1965).

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