35
Clinica C~~mica Acta, 59 (1975) 35-39 @ Ekevier Scientific Publishing Company, Amsterdam - Printed in the Netherlands
CCA 6779
DETERMINATION OF PHOSPHATE IN SERUM AND URINE BY A SINGLE STEP MALACHITE-GREEN METHOD
ANDERS KALLNER Department of Clinical Chemistry, Hospital, Stockholm (Sweden)
Karolinsha
Institute
at the Serafimer
(Received August 6, 1974)
The highly sensitive malachite-green method for the determination of phosphate requires a protective colloid in order to avoid precipitation of the formed dye salt. A polyvinylalcohol proved to be suitable and permit the determination of phosphate in urine and in serum without prior precipitation of the proteins. The present method is suitable for a one-step procedure which allows the analysis of several hundred samples per man-hour and is applicable to microquantities of serum or urine.
Introduction Phosphate ion liberates a strongly coloured compound from a malachitegreen molybdate complex in acid medium. This reaction has been used for the quantitative de~rmination of phosphors in serum after the precipi~tion of proteins [l] and in water [Z] . The sensitivity of methods for the determination of phosphate, based on the formation of the malachite-green complex, is claimed to be about 30 times higher than for the widely used Fiske-SubbaRow method. The method has been improved [3] and modified in order to avoid the precipitation of protein [4]. In our hands, the latter method was irreproducible, probably because the addition of molybdate had been omitted from the description. This failure prompted us to reinvestigate the reaction in order to make it possible to add reagents in one step, thus achieving a rational workflow. Materials and Methods In the preparation of reagents, bidistilled water was used and all glassware was acid-washed and well rinsed with bidistilled water. Reagents were of analytical grade.
36
Reagents Urea solution 5 mol/l solution
(300 g/l).
(a) Malachite-green oxalate (e.g. Merck 13983, 700 mg (0.75 mmoi), is dissolved in about 1 1 water. (b) Ammonium molybdate (e.g. Mallinckrodt 3420), 24.7 g (20.0 mmol) (paramolybdate (NH4 )6 Mo7 0, 4 . 4 HZ 0). is dissolved in 500 ml HCl (6 moljl). (c) Polyvinylalcohol (eg. Polyviol V 03120 WACKER-CHEMIE GmBH; MW c:a 1300), 10 g, is dissolved in about 100 ml boiling water. After cooling, a, b, and c are poured together and diluted to 3.0 1 with water. The reagent can be used as soon as it has reached a stable yellow-brown colour which takes about 30 minutes. A preparation of this reagent can be used for at least one month when stored in the dark. A slight precipitation is sometimes formed on long standing; however, the clear supernatant can be decanted and used. working reagent A working reagent is prepared by 4 parts of the malachite-green and 1 part of urea solution. This reagent should be made immediately use and is stable for at least one day. Standard
reagent prior to
solutions
Stock standard Potassium
dihydrogenphosphate,
32.5 mmof/l
(4.422 g/l).
Working standards Standard for urine, 0.650 mmol/l: Stock standard, 1 ml, is diluted to 50 ml with water. Standard for serum, 0.650 mmol/l: Stock standard, 1 ml, is diluted to 50 ml with dialyzed serum. The diluent was prepared by dialyzing pooled serum overnight against water and cleared by the addition of 0.9 g sodium chloride. The absence of phosphate in the diluent was checked before use (Fig. I). Procedure Working reagent, 2.5 ml, is added to 0.02 ml of the sample. Alternatively, 2.0 ml of malachite-green and 0.5 ml of urea solution are simultaneously added to a 0.02-ml sample by means of a suitable automatic pipette. A greenish colour immediately starts developing and the reaction continues at somewhat different rates for different test solutions (Fig. 2). After 60 minutes, the absorbance is read at 630 nm. Urine samples could be read already after 20-30 minutes. Using a fast pipetting system and a suitable spectrometer, several hundred samples can be analyzed per working hour.
37
Fig. 1. Decay
of phosphate
Fig. 2. Development and dialyzed serum.
concentration
of colour reaction Room temperature,
during dialyzes of phosphate 630 nm.
of serum against water.
in, from
above:
urine.
water.
5% albumin.
serum.
Calculation Standard curves of phosphate in dialyzed serum and in water are shown in Fig. 3. Within the physiological ranges the correlation between absorbance and concentration approaches a straight line passing through the origin. This permits the use of only one reference point to achieve an acceptable standard curve and a factorizing spectrometer could be used. Since the phosphate concentration of urine is high and varies greatly, it is advisable to dilute urine 20-40 times before the addition of the colour reagent. Recoveries were checked by the addition of a concentrated phosphate solution to a dialyzed serum and to a sample from a pooled serum (Table I). Results and Discussion Liberation of the greenish colour is enhanced by the presence of urea. The intensity of the developed colour increased continuously on the addition of urea
10 Fig. 3. (*-)
30
mmolil
Absorbance of standard versus concentration.
solutions
of phosphate
in dialyzed
serum
( AA)
and
in water
38
TABLE
I
ADDITION SAMPLE
OF
PHOSPHATE
IN
\VATER
TO
DIALYZED
SERUM
(A)
AND
TO
A POOLED
SERUM
(H)
B
A Calculated
Found
Rcvowry
Calculated
Found
Krcm
(mmolil)
(mm
Clinica C~~mica Acta, 59 (1975) 35-39 @ Ekevier Scientific Publishing Company, Amsterdam - Printed in the Netherlands
CCA 6779
DETERMINATION OF PHOSPHATE IN SERUM AND URINE BY A SINGLE STEP MALACHITE-GREEN METHOD
ANDERS KALLNER Department of Clinical Chemistry, Hospital, Stockholm (Sweden)
Karolinsha
Institute
at the Serafimer
(Received August 6, 1974)
The highly sensitive malachite-green method for the determination of phosphate requires a protective colloid in order to avoid precipitation of the formed dye salt. A polyvinylalcohol proved to be suitable and permit the determination of phosphate in urine and in serum without prior precipitation of the proteins. The present method is suitable for a one-step procedure which allows the analysis of several hundred samples per man-hour and is applicable to microquantities of serum or urine.
Introduction Phosphate ion liberates a strongly coloured compound from a malachitegreen molybdate complex in acid medium. This reaction has been used for the quantitative de~rmination of phosphors in serum after the precipi~tion of proteins [l] and in water [Z] . The sensitivity of methods for the determination of phosphate, based on the formation of the malachite-green complex, is claimed to be about 30 times higher than for the widely used Fiske-SubbaRow method. The method has been improved [3] and modified in order to avoid the precipitation of protein [4]. In our hands, the latter method was irreproducible, probably because the addition of molybdate had been omitted from the description. This failure prompted us to reinvestigate the reaction in order to make it possible to add reagents in one step, thus achieving a rational workflow. Materials and Methods In the preparation of reagents, bidistilled water was used and all glassware was acid-washed and well rinsed with bidistilled water. Reagents were of analytical grade.
36
Reagents Urea solution 5 mol/l solution
(300 g/l).
(a) Malachite-green oxalate (e.g. Merck 13983, 700 mg (0.75 mmoi), is dissolved in about 1 1 water. (b) Ammonium molybdate (e.g. Mallinckrodt 3420), 24.7 g (20.0 mmol) (paramolybdate (NH4 )6 Mo7 0, 4 . 4 HZ 0). is dissolved in 500 ml HCl (6 moljl). (c) Polyvinylalcohol (eg. Polyviol V 03120 WACKER-CHEMIE GmBH; MW c:a 1300), 10 g, is dissolved in about 100 ml boiling water. After cooling, a, b, and c are poured together and diluted to 3.0 1 with water. The reagent can be used as soon as it has reached a stable yellow-brown colour which takes about 30 minutes. A preparation of this reagent can be used for at least one month when stored in the dark. A slight precipitation is sometimes formed on long standing; however, the clear supernatant can be decanted and used. working reagent A working reagent is prepared by 4 parts of the malachite-green and 1 part of urea solution. This reagent should be made immediately use and is stable for at least one day. Standard
reagent prior to
solutions
Stock standard Potassium
dihydrogenphosphate,
32.5 mmof/l
(4.422 g/l).
Working standards Standard for urine, 0.650 mmol/l: Stock standard, 1 ml, is diluted to 50 ml with water. Standard for serum, 0.650 mmol/l: Stock standard, 1 ml, is diluted to 50 ml with dialyzed serum. The diluent was prepared by dialyzing pooled serum overnight against water and cleared by the addition of 0.9 g sodium chloride. The absence of phosphate in the diluent was checked before use (Fig. I). Procedure Working reagent, 2.5 ml, is added to 0.02 ml of the sample. Alternatively, 2.0 ml of malachite-green and 0.5 ml of urea solution are simultaneously added to a 0.02-ml sample by means of a suitable automatic pipette. A greenish colour immediately starts developing and the reaction continues at somewhat different rates for different test solutions (Fig. 2). After 60 minutes, the absorbance is read at 630 nm. Urine samples could be read already after 20-30 minutes. Using a fast pipetting system and a suitable spectrometer, several hundred samples can be analyzed per working hour.
37
Fig. 1. Decay
of phosphate
Fig. 2. Development and dialyzed serum.
concentration
of colour reaction Room temperature,
during dialyzes of phosphate 630 nm.
of serum against water.
in, from
above:
urine.
water.
5% albumin.
serum.
Calculation Standard curves of phosphate in dialyzed serum and in water are shown in Fig. 3. Within the physiological ranges the correlation between absorbance and concentration approaches a straight line passing through the origin. This permits the use of only one reference point to achieve an acceptable standard curve and a factorizing spectrometer could be used. Since the phosphate concentration of urine is high and varies greatly, it is advisable to dilute urine 20-40 times before the addition of the colour reagent. Recoveries were checked by the addition of a concentrated phosphate solution to a dialyzed serum and to a sample from a pooled serum (Table I). Results and Discussion Liberation of the greenish colour is enhanced by the presence of urea. The intensity of the developed colour increased continuously on the addition of urea
10 Fig. 3. (*-)
30
mmolil
Absorbance of standard versus concentration.
solutions
of phosphate
in dialyzed
serum
( AA)
and
in water
38
TABLE
I
ADDITION SAMPLE
OF
PHOSPHATE
IN
\VATER
TO
DIALYZED
SERUM
(A)
AND
TO
A POOLED
SERUM
(H)
B
A Calculated
Found
Rcvowry
Calculated
Found
Krcm
(mmolil)
(mm
