ANALYTICAL BIOCHEMISTRY 154,420-423 (1986)

Determination of Phosphorus in Cereal Lipids

MIKLOS I. P. KOVACS

Received September 24. 1985

The effect of digestion methods on the determination of phosphorus in cereal lipids was rein- vestigated. Samples were either digested with sulfuric acid or ashed in a muffle furnace at 600°C. The standard deviation and the coefficient of variation were significantly higher for the acid- digested samples. Ashing gave more reliable results, especially when large amounts of lipid material had to be oxidized. ID 1986 Academx Press. Inc.

KEY WORDS: phosphorus: determination: lipids: cereal.

The numerous publications concerning the calorimetric determination of phosphorus in biological materials reflects the difficulties in- herent in the analysis (l-4). The accuracy of its determination depends almost entirely upon the oxidation or digestion of the organic material. Samples containing complex lipids, such as cereal lipids. are especially difficult to oxidize. Once the organic material is oxidized and the phosphorus is converted to inorganic form, the determination of phosphorus is a relatively simple procedure.

Basically there are two different methods commonly used to oxidize organic matter for phosphorus determination: wet oxidation and dry oxidation.

There are several publications describing wet oxidation (l-4). The sample, containing organic phosphate, is heated in the presence of strong acid(s) such as perchloric, sulfuric, and nitric acids. or their combinations. It was shown that acid concentration is critical to obtain reproducible determinations of phos- phorus in phospholipids (5). Using the wet digestion method, prolonged heating is nec- essary if large amounts of organic matter have to be digested to liberate inorganic phosphate. This leads to loss of acidity and consequently loss of reproducibility if the acid concentration is not adjusted within a narrow range (5). Be- cause phosphorus is determined colorimetri- tally, acid digestion normally includes the ad-

dition of hydrogen peroxide to remove inter- fering color prior to color development (6). The hydrogen peroxide. however. has to be completely destroyed by further heating and addition of sulfite (4) to avoid its interference with the formation of molybdenum blue (7). Further, it has been reported that during mo- lybdenum blue color formation the required range of acid concentration is different at dif- ferent temperatures and heating times (4). Optimum acid concentration and temperature of the color reaction are also influenced by the kind of acid used in the wet oxidation pro- cess (5).

In dry digestion, the sample is oxidized in a furnace at about 600°C in the presence of heavy metal salt to avoid volatilization of or- ganic phosphate (9).

Recently different methods for the deter- mination of phosphorus of different origins have been compared, and dry digestion was found to be the most accurate (8).

This study describes an accurate and simple method for the determination of phosphorus in samples containing complex cereal lipids using dry digestion.

MATERIALS AND METHODS

Muterials

All reagents were of analytical grade. Sul- furic acid (sp grav 1.84, 96’%), ammonium

0003-2697/86 $3.00 Copyright 8 1986 by Academic Press. Inc All ngbts of reproduction in any form reserved.

4’0

DETERMINATION OF PHOSPHORUS IN CEREAL LIPIDS 421

TABLE 1

RECOVERY OF PHOSPHORUS FROM WHEAT LIPIDS USING DRY DIGESTION TECHNIQUE

Wheat hpid Added LPC Total P

CLE P analyzed’ P calculatedb Calculated b Analyzed’ Recovery b-%) m (Pa beg) (P!z) m’u)

128 0.8 1.0 1.8 1.8 100 256 1.5 2.0 3.5 3.6 103 512 3.1 4.0 7.1 7.3 103 768 4.5 6.0 10.5 10.6 101

1024 6.1 8.0 14.1 14.1 100 1280 7.8 10.0 17.8 17.6 99

’ Analyzed P based on standard curve of inorganic phosphate. ’ Calculated P based on the phosphate content (theoretical) of Iysophosphatidylcholine.

molybdate (IVH4)6M07024 * 4H20) and L-( +)-

ascorbic acid were obtained from BDH chem- icals. L-Lu-Lysophosphatidylcholine (LPC)’ was obtained from Sigma Chemical Company (St. Louis, MO.). All solvents were of reagent grade from Fisher Scientific Company (Pittsburgh, Pa.). Crude lipid extracts (CLE) were prepared from a Canadian hard red spring wheat flour by extracting with water-saturated n-butanol (Morrison, 1964).

Ifi>t di,qc~tiorz. The wet digestion was per- formed according to Morrison (4).

Dq, digestion. Extracts (0. l-l ml) contain- ing I-20 pg o.f phosphorus were transferred to crucibles (crucibles have to be in good con- dition to avoid strong absorption of the resi- due). The sample solvents were evaporated to almost dryness and 1 ml of 10% zinc acetate was added to the crucible. ensuring coverage of all the sample residue. Prior to dry digestion the crucibles were placed into a water bath or onto a hot plate to dry. When using the hot plate the temperature setting was low to avoid splashing from boiling. The dried samples were heated further on the hot plate until fuming stopped (about 5 min), and then the crucibles

’ Abbreviations used: LPC, L-a-Lysophosphatidylcho- line: CLE, crude lipid extracts.

were transferred into a muffle furnace at a temperature of 550-600°C for 2 h.

The color reaction was basically that of Ki Soon Rhee and Dugan (5) with the following modifications. After the crucibles were re-

li

J ‘0

FIG. 1. Standard curves for inorganic phosphate deter- minations: inorganic phosphate (0). crude lipid extract (0). and dry digested crude lipid extracts without zinc ac- etate (A)

42’ MIKLOS I.

1,4r---

FIG. 2. Effect of acid concentration on phosphorus color development (0) and pH (0).

moved from the furnace and cooled to room temperature, 1 ml of 3% molybdate in 5 N sulfuric acid and 6.5 ml of 1% ascorbic acid solutions were added to the residue of the dry digestion (total volume was 7.5 ml). Imme- diately the mixture was well mixed with a glass rod or magnetic stirring bar, and an aliquot of about 5 ml was transferred into test tubes. The tubes then were heated in a boiling water bath for 5 min and cooled to room tempera- ture, and the absorbance read at 820 nm against the blank prepared without sample. The absorbance of the samples was stable for at least 48 h.

The pH was determined using an aliquot of each sample.

P. KOVACS

Rrcm~er~~ .st~dirs. Recovery of phosphorus was evaluated by adding known amounts ot LPC to the samples prior to digestion.

A standard curve for the calorimetric de- termination was prepared using inorganic phosphate.

RESULTS AND DISCUSSION

Results of the recovery of phosphorus from wheat lipids and added lysophosphatidylcho- line, using the dry digestion method. are pre- sented in Table 1. The percentage recovery. obtained by adding varying amounts of LPC (with a known amount of phosphorus) to CLE, shows that when using dry digestion there is no loss of phosphorus resulting from ashing and/or from color reactions. Similar conclu- sions can be obtained from Fig. 1 when the standard curve. obtained by using inorganic phosphate (no digestion) and crude lipid ex- tract values (dry digestion), are compared. The importance of zinc acetate is also illustrated in Fig. 1. When samples were digested without zinc acetate, a considerable amount of phos- phorus was lost, probably due to evaporation of phospholipids by the high digestion tem- perature.

The optimum acid concentration for the color reaction is critical and lies between 0.4 and 0.7 N (Fig. 2). similar to that reported by Ki Soon Rhee and Dugan (5). but the range of pH was, however, lower: I .5-0.7 instead of 4.0-3.8.

TABLE 2

STATISTICAL EVALUATION OF WET AND DRY DIGESTION METHODS FOR THE DETERMINATION OF PHOSPHORUS IN CLE

Total Wet digestion Dry digestion CLE Added LPC calculated (md p (/G) p (Pd .\-” Zh CYb: I” STh ix‘

0.8 - 4.70 4.9 0.41 8.4 4.71 0.15 3.2 1.6 9.40 Il.3 0.47 4.2 9.35 0.2 1 2.2

0.8 4 8.7 I 10.5 1.90 18.1 8.91 0.16 1.8 1.6 8 17.35 21.78 5.05 23.2 17.03 0.32 1.9

y Mean value. ’ Standard deviation. ” Coefficient of variation B.

DETERMINATION OF PHOSPHORUS IN CEREAL LIPIDS 423

Statistical evaluation of the two digestion methods is presented in Table 2. The corre- lation between added and recovered phospho- rus is better when dry digestion was used. The standard deviation and the coefficient of vari- ation percentage are significantly higher for the wet digestion method. The higher phos- phorus content obtained by wet digestion is probably due to the higher acid concentration resulting from evaporation of acid by excessive heating, as compared to that used for the preparation of standard curve. Consequently. when wet digestion is used for samples difficult to digest. the acid concentration has to be ad- justed to avoicl errors. This, however, requires some experience and is also time consuming.

Dry digestion eliminates the use of danger- ous concentrated acids and consequently the problem of controlling reagent concentrations. The phosphorus residue after digestion is usually dissolved in concentrated sulfuric acid (8). However, there were no differences in the results when I or 5 N sulfuric acid was used to dissolve the residue after dry digestion.

The wet digestion method gave satisfactory

standard curves when using inorganic phos- phate, indicating that the decomposition of samples is the most critical step (8). This study indicates that dry digestion of the samples gives more reliable results. especially when samples difficult to digest, such as cereal lipids, are used. Moreover, the dry digestion method re- quires less labor.

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