- Email: [email protected]
Flow-injection determination of creatine in animal tissues
Analytica Chimica Acta, 208 (1988) 307-312 Elsevier Science Publishers B.V., Amsterdam -
307 Printed in The Netherlands
Short Communication
FLOW-INJECTION ANIMAL TISSUES
DETERMINATION
OF CREATINE IN
ZDENfiK DVORAK Meat Industry Research Institute, 61200 Brno (Czechoslovakia) (Received 15th March 1987)
Summary. After deproteination of samples with trichloroacetic acid, creatine is determined by reaction with 1-naphthol and biacetyl, based on a stopped-flow method. The calibration graph is linear over the range O-250 mg l-r, and recoveries from muscle samples are quantitative.
Determination of creatine is of interest in clinical work during studies of muscle and kidney disorders. It is also of interest in food science because the concentration of creatine in meat of slaughtered animals appears to be directly related to the muscle protein content, free of connective tissue proteins [ 11. Therefore, creatine can serve as an indicator of meat quality. There are two basic methods for the determination of creatine. One exploits the possibility of converting creatine to creatinine which is determined by the Jaffe reaction with sodium picrate [ 21. The other is based on the reaction of creatine with 1-naphthol and biacetyl [ 3,4]. Compounds interfering in the Jaffe reaction can be separated by absorption on Lloyd’s reagent [5] or by elution through strongly acidic cation-exchangers [ 61. The latter procedure has also been used for the 1-naphthol/biacetyl reaction [ 71. The Jaffe method for creatinine has been modified for use in an air-segmented flow analyzer [ 81. Flow injection analysis has also been used with this reaction [ 91. The air-segmented flow procedure has also been applied for the determination of creatine by the 1-naphthol/biacetyl reaction [lo]. Adaptation of this method for flow injection analysis is described below. Experimental Apparatus. A FIAStar 5020 analyzer was used in combination with a Chemifold II, FIAStar 5022 detector/controller and FIAStar 5023 spectrophotometer (all Tecator, Sweden). A potentiometric chart recorder type OH-814/l (Radelkis, Budapest) was used for recording peaks. Details of the manifold are given in Fig. 1.
Fig. 1. Manifold for the determination of creatine in the FIAStar with Chemifold II: S, sample, valve loop size 200 pL1;P, peristaltic pump; C, carrier stream; R,, Rz, reagent streams; W, waste. Pump tube volumes are given in ml m-l; for mixing coils, length is in cm and i.d. in mm.
Reagents. All chemicals used were of analytical-reagent grade. Solutions were prepared with water redistilled from a glass apparatus, and before use were filtered and degassed. A standard creatine solution was prepared by weighing 56.9 mg of creatine monohydrate (Merck), dissolving it in trichloroacetic acid solution (30 g 1-l) and diluting to 100 ml with the trichloroacetic acid solution. This solution was further diluted with trichloroacetic acid solution to give 50, 25 and 12.5 mg 1-l creatine solutions. All creatine solutions were freshly prepared. The 30 g 1-l trichloroacetic acid solution was also used as the carrier stream. This concentration appeared to be satisfactory for precipitating the protein from samples, and also for minimizing the rate of cyclization of creatine to creatinine. 1-Naphthol reagent(R1) (20 g 1-l) was prepared by dissolving lnaphthol in an alkaline solution (60 g of NaOH and 160 g of anhydrous sodium carbonate in 11 of water). The solution was prepared daily. The biacetyl stock solution was 10 g 1-l in water. Before use, 1 ml was diluted with 19 ml of water to provide the R2 reagent. Sample preparation. Animal tissues, especially muscles of slaughtered animals must be sampled carefully to obtain a representative sample. After coarse cutting of the muscle and mixing, about 250 g of tissue was homogenized in a Moulinex mill. A weighed portion (10.0 g) of the paste was then homogenized in a Warring blender with 200 ml of the trichloroacetic acid solution. After filtration of precipitated protein and fat, the colourless extract was used for analysis on the same day. Manual method for creutine determination. An aliquot (1 ml) of sample solution or standard solution was pipetted into a calibrated tube and diluted with water to 14 ml. After mixing, 4.0 ml of the alkaline solution of 1-naphthol and 2.0 ml of the diluted biacetyl solution were added. The absorbance of the red solution was measured at 520 nm after 20 min. Recouery. For recovery determination, filtered extracts of eight different cuts of beef were diluted with equal volumes of standard solutions containing crea-
309
tine (25 or 100 mg 1-l). For the determination of creatine in these solutions, the following times were used (see below): t1= 19 s, t2= 10 s, tinj= 20 s. Results and discussion The manual method yields a linear calibration graph at least up to 250 lugof ‘creatine. Nevertheless, it was not possible to detect any absorbance peak in a flow-injection procedure as in Fig. 1. An increase of the reaction time, however, proved to be effective. Instead of lengthening the mixing coil, the stopped-flow method was used. Subsequently the optimum flow-time after injection of the sample ( tl) and the stopped-flow time ( tz) for achieving greatest sensitivity were evaluated, tinjis the time used to wash out the injection valve. The changes in peak height with flow time ( t1) are shown in Fig. 2. At tl = lo15 s, two peaks are obtained; the smaller peak is recorded by the FIAStar but the following peak, obtained just after interruption of the stopped flow, which is usually higher, is not. This phenomenon is best seen for ti = 15 s. The double maxima suggest that at the time of stopped flow, dispersion was incomplete within the sample zone until ti--20 s. Thus the time 22 s can be regarded as optimal for stopping the flow. The influence of stopped-flow time (t2) is shown in Fig. 3. The sensitivity increases with increasing t2 and linear calibration graphs are obtained with tinj= 20 S. The non-linear plots obtained with tinj= 10 s indicate that this washout time is inadequate for complete removal of the sample from the injection
Fig. 2. Effect of flow time on peaks for 50 mg l- 1creatine. given below each peak.
tinj = 20 s, tz = 20
S; tl
values (in
s)
are
310
valve. The positive intercepts on the concentration axis are probably related to the total time needed for the creatine reaction. Figure 3 shows clearly that the sensitivity of the method can be changed by adjusting t2. The influence of flow-rates was investigated from the aspect of neutralizing the acidic carrier stream (C) and sample by the alkaline reagent RI, and providing enough alkalinity for reaction with 1-naphthol. The pump tubing shown in Fig. 1 satisfied this requirement. Table 1 shows that the flow rate of the
900
800 700
I
600 1 0 & vl 500-
400 -
300 -
._ - -~ -
-I
150 CREATINE
200 (mg
250
I-’ )
lOmln
Fig. 3. Dependence of peak-height signals on creatine concentration and t2 (given in s on each curve): (0) t,=t,,=20s; (0) t,=20s,ti,j=10s. Fig. 4. Responses for a series of creatine solutions: (I-V) standard creatine solutions, 50,25, 12.5, 7.5 and 2.5 mg 1-l; (l-10) creatine solutions from different cuts of meat. (tinj=20 s, t, =22 s, t2=20 s.) TABLE 1 Influence of flow rate of the biacetyl stream (R,) on peak heights” Creatine concentration (mg 1-l) Signal with R, at 0.42 Signal with R2 at 0.23
10 34 80
30 216 363
50 411 647
“Other conditions: pump tubes for R, and C both 0.42 ml m-l; t, = 15 S,
tz =
30 S,
ti,
=
20 S.
311
biacetyl stream (& ) also affected the sensitivity; the pump tube with a volume of 0.23 ml m-l gave a greater sensitivity than the larger tube. Attempts to pool reagents R, and R2 into one solution were not successful. Peaks for quadruplicate injections of ten different cuts of pork are shown in Fig. 4. The data are evaluated in Table 2, where the results are compared with average creatine contents determined by the manual method. The mean relative error of the flow method was 1.67%. The variability of the results was not dependent on the creatine content in the sample, and no effect of carry-over was observed. Comparison of the results of the new method with the manual method indicates good agreement. Recoveries of creatine added to eight different muscle samples at two levels (25 and 100 mg 1-l) were 103.6 2 2.3% and 99.9 ? 2.1%, respectively. The following compounds (0.5 mmol 1-l) were tested for reactivity with lnaphthol and biacetyl: creatinine, arginine hydrochloride, guanidine hydrochloride, methionine, sodium pyruvate, sodium glycerophosphate, cr-oxoglutaric acid, urea, bilirubin and ascorbic acid. Each compound was dissolved in a 30 g 1-l solution of trichloroacetic acid which contained 0.5 mmol 1-l creatine. If the reactivity of creatine is taken as lOO%, the only reactions observed were with methionine (11.0% ), arginine hydrochloride (13.5%) and guanidine hydrochloride (45.0% ). The last is not found in animal tissues and consequently cannot interfere in analyses of material of animal origin. The other compounds tested did not react and also did not inhibit the creatine reaction. The method was used in routine analyses of different cuts of beef and pork. TABLE 2 Results for creatine determination
in meat samples (peaks shown in Fig. 3)
Sample no.
method
1 2 3 4 5 6 I 8 9 10 Mean
Flow-injection 2” (g kg-‘)
R.s.d. (%)
4.27 3.92 3.28 3.55 2.58 1.97 1.20 4.24 3.56 3.87
0.36 0.15 0.79 0.48 1.51 0.66 0.33 0.31 0.36 0.31 0.43
Manual method fb (gkg-‘1
Recovery’ (%I
4.30 3.81 3.32 3.48 2.54 1.90 1.27 4.19 3.46 3.84
100.8 97.2 101.2 98.0 98.5 96.3 105.8 98.9 97.1 99.2 99.3
“Mean creatine concentration, four measurements. bMean of four measurements. “Ratio of manual to flow-injection result.
312
It can be applied to any animal tissue. In the skeletal muscle, the creatine content is high and corresponds to the net content of muscle proteins. By adjusting the stopped-flow time ( tz) creatine can be determined over a wide range of concentrations.
REFERENCES 1 2 3 4 5 6 I 8 9 10
Z. Dvoi& J. Sci. Food Agric., 32 (1981) 1033. J.W. Dubnoff, in S.P. Colowick and N.O. Kaplan (Eds.), Methods in Enzymology, Vol. 3, Academic, New York, 1957, p. 637. M.M. Barrett, J. Pathol. Bacterial., 42 (1936) 441. P. Eggleton, S.R. Elsden and N. Gough, Biochem. J., 37 (1943) 526. H. Borsook, J. Biol. Chem., 110 (1935) 481. E.P. Diamandis and T.P. Hadjiioannou, Clin. Chem., 27 (1981) 455. E. Mussini, L. Colombo, G. de Ponte and F. Marcucci, J. Chromatogr., 305 (1984) 450. E.R. Ryan and W.H.C. Walker, Microchem. J., 24 (1980) 500. J.F. van Staden, Fresenius’ Z.Anal. Chem., 315 (1983) 141. W.J. Griffiths, Clin. Chim. Acta, 9 (1964) 210.
307 Printed in The Netherlands
Short Communication
FLOW-INJECTION ANIMAL TISSUES
DETERMINATION
OF CREATINE IN
ZDENfiK DVORAK Meat Industry Research Institute, 61200 Brno (Czechoslovakia) (Received 15th March 1987)
Summary. After deproteination of samples with trichloroacetic acid, creatine is determined by reaction with 1-naphthol and biacetyl, based on a stopped-flow method. The calibration graph is linear over the range O-250 mg l-r, and recoveries from muscle samples are quantitative.
Determination of creatine is of interest in clinical work during studies of muscle and kidney disorders. It is also of interest in food science because the concentration of creatine in meat of slaughtered animals appears to be directly related to the muscle protein content, free of connective tissue proteins [ 11. Therefore, creatine can serve as an indicator of meat quality. There are two basic methods for the determination of creatine. One exploits the possibility of converting creatine to creatinine which is determined by the Jaffe reaction with sodium picrate [ 21. The other is based on the reaction of creatine with 1-naphthol and biacetyl [ 3,4]. Compounds interfering in the Jaffe reaction can be separated by absorption on Lloyd’s reagent [5] or by elution through strongly acidic cation-exchangers [ 61. The latter procedure has also been used for the 1-naphthol/biacetyl reaction [ 71. The Jaffe method for creatinine has been modified for use in an air-segmented flow analyzer [ 81. Flow injection analysis has also been used with this reaction [ 91. The air-segmented flow procedure has also been applied for the determination of creatine by the 1-naphthol/biacetyl reaction [lo]. Adaptation of this method for flow injection analysis is described below. Experimental Apparatus. A FIAStar 5020 analyzer was used in combination with a Chemifold II, FIAStar 5022 detector/controller and FIAStar 5023 spectrophotometer (all Tecator, Sweden). A potentiometric chart recorder type OH-814/l (Radelkis, Budapest) was used for recording peaks. Details of the manifold are given in Fig. 1.
Fig. 1. Manifold for the determination of creatine in the FIAStar with Chemifold II: S, sample, valve loop size 200 pL1;P, peristaltic pump; C, carrier stream; R,, Rz, reagent streams; W, waste. Pump tube volumes are given in ml m-l; for mixing coils, length is in cm and i.d. in mm.
Reagents. All chemicals used were of analytical-reagent grade. Solutions were prepared with water redistilled from a glass apparatus, and before use were filtered and degassed. A standard creatine solution was prepared by weighing 56.9 mg of creatine monohydrate (Merck), dissolving it in trichloroacetic acid solution (30 g 1-l) and diluting to 100 ml with the trichloroacetic acid solution. This solution was further diluted with trichloroacetic acid solution to give 50, 25 and 12.5 mg 1-l creatine solutions. All creatine solutions were freshly prepared. The 30 g 1-l trichloroacetic acid solution was also used as the carrier stream. This concentration appeared to be satisfactory for precipitating the protein from samples, and also for minimizing the rate of cyclization of creatine to creatinine. 1-Naphthol reagent(R1) (20 g 1-l) was prepared by dissolving lnaphthol in an alkaline solution (60 g of NaOH and 160 g of anhydrous sodium carbonate in 11 of water). The solution was prepared daily. The biacetyl stock solution was 10 g 1-l in water. Before use, 1 ml was diluted with 19 ml of water to provide the R2 reagent. Sample preparation. Animal tissues, especially muscles of slaughtered animals must be sampled carefully to obtain a representative sample. After coarse cutting of the muscle and mixing, about 250 g of tissue was homogenized in a Moulinex mill. A weighed portion (10.0 g) of the paste was then homogenized in a Warring blender with 200 ml of the trichloroacetic acid solution. After filtration of precipitated protein and fat, the colourless extract was used for analysis on the same day. Manual method for creutine determination. An aliquot (1 ml) of sample solution or standard solution was pipetted into a calibrated tube and diluted with water to 14 ml. After mixing, 4.0 ml of the alkaline solution of 1-naphthol and 2.0 ml of the diluted biacetyl solution were added. The absorbance of the red solution was measured at 520 nm after 20 min. Recouery. For recovery determination, filtered extracts of eight different cuts of beef were diluted with equal volumes of standard solutions containing crea-
309
tine (25 or 100 mg 1-l). For the determination of creatine in these solutions, the following times were used (see below): t1= 19 s, t2= 10 s, tinj= 20 s. Results and discussion The manual method yields a linear calibration graph at least up to 250 lugof ‘creatine. Nevertheless, it was not possible to detect any absorbance peak in a flow-injection procedure as in Fig. 1. An increase of the reaction time, however, proved to be effective. Instead of lengthening the mixing coil, the stopped-flow method was used. Subsequently the optimum flow-time after injection of the sample ( tl) and the stopped-flow time ( tz) for achieving greatest sensitivity were evaluated, tinjis the time used to wash out the injection valve. The changes in peak height with flow time ( t1) are shown in Fig. 2. At tl = lo15 s, two peaks are obtained; the smaller peak is recorded by the FIAStar but the following peak, obtained just after interruption of the stopped flow, which is usually higher, is not. This phenomenon is best seen for ti = 15 s. The double maxima suggest that at the time of stopped flow, dispersion was incomplete within the sample zone until ti--20 s. Thus the time 22 s can be regarded as optimal for stopping the flow. The influence of stopped-flow time (t2) is shown in Fig. 3. The sensitivity increases with increasing t2 and linear calibration graphs are obtained with tinj= 20 S. The non-linear plots obtained with tinj= 10 s indicate that this washout time is inadequate for complete removal of the sample from the injection
Fig. 2. Effect of flow time on peaks for 50 mg l- 1creatine. given below each peak.
tinj = 20 s, tz = 20
S; tl
values (in
s)
are
310
valve. The positive intercepts on the concentration axis are probably related to the total time needed for the creatine reaction. Figure 3 shows clearly that the sensitivity of the method can be changed by adjusting t2. The influence of flow-rates was investigated from the aspect of neutralizing the acidic carrier stream (C) and sample by the alkaline reagent RI, and providing enough alkalinity for reaction with 1-naphthol. The pump tubing shown in Fig. 1 satisfied this requirement. Table 1 shows that the flow rate of the
900
800 700
I
600 1 0 & vl 500-
400 -
300 -
._ - -~ -
-I
150 CREATINE
200 (mg
250
I-’ )
lOmln
Fig. 3. Dependence of peak-height signals on creatine concentration and t2 (given in s on each curve): (0) t,=t,,=20s; (0) t,=20s,ti,j=10s. Fig. 4. Responses for a series of creatine solutions: (I-V) standard creatine solutions, 50,25, 12.5, 7.5 and 2.5 mg 1-l; (l-10) creatine solutions from different cuts of meat. (tinj=20 s, t, =22 s, t2=20 s.) TABLE 1 Influence of flow rate of the biacetyl stream (R,) on peak heights” Creatine concentration (mg 1-l) Signal with R, at 0.42 Signal with R2 at 0.23
10 34 80
30 216 363
50 411 647
“Other conditions: pump tubes for R, and C both 0.42 ml m-l; t, = 15 S,
tz =
30 S,
ti,
=
20 S.
311
biacetyl stream (& ) also affected the sensitivity; the pump tube with a volume of 0.23 ml m-l gave a greater sensitivity than the larger tube. Attempts to pool reagents R, and R2 into one solution were not successful. Peaks for quadruplicate injections of ten different cuts of pork are shown in Fig. 4. The data are evaluated in Table 2, where the results are compared with average creatine contents determined by the manual method. The mean relative error of the flow method was 1.67%. The variability of the results was not dependent on the creatine content in the sample, and no effect of carry-over was observed. Comparison of the results of the new method with the manual method indicates good agreement. Recoveries of creatine added to eight different muscle samples at two levels (25 and 100 mg 1-l) were 103.6 2 2.3% and 99.9 ? 2.1%, respectively. The following compounds (0.5 mmol 1-l) were tested for reactivity with lnaphthol and biacetyl: creatinine, arginine hydrochloride, guanidine hydrochloride, methionine, sodium pyruvate, sodium glycerophosphate, cr-oxoglutaric acid, urea, bilirubin and ascorbic acid. Each compound was dissolved in a 30 g 1-l solution of trichloroacetic acid which contained 0.5 mmol 1-l creatine. If the reactivity of creatine is taken as lOO%, the only reactions observed were with methionine (11.0% ), arginine hydrochloride (13.5%) and guanidine hydrochloride (45.0% ). The last is not found in animal tissues and consequently cannot interfere in analyses of material of animal origin. The other compounds tested did not react and also did not inhibit the creatine reaction. The method was used in routine analyses of different cuts of beef and pork. TABLE 2 Results for creatine determination
in meat samples (peaks shown in Fig. 3)
Sample no.
method
1 2 3 4 5 6 I 8 9 10 Mean
Flow-injection 2” (g kg-‘)
R.s.d. (%)
4.27 3.92 3.28 3.55 2.58 1.97 1.20 4.24 3.56 3.87
0.36 0.15 0.79 0.48 1.51 0.66 0.33 0.31 0.36 0.31 0.43
Manual method fb (gkg-‘1
Recovery’ (%I
4.30 3.81 3.32 3.48 2.54 1.90 1.27 4.19 3.46 3.84
100.8 97.2 101.2 98.0 98.5 96.3 105.8 98.9 97.1 99.2 99.3
“Mean creatine concentration, four measurements. bMean of four measurements. “Ratio of manual to flow-injection result.
312
It can be applied to any animal tissue. In the skeletal muscle, the creatine content is high and corresponds to the net content of muscle proteins. By adjusting the stopped-flow time ( tz) creatine can be determined over a wide range of concentrations.
REFERENCES 1 2 3 4 5 6 I 8 9 10
Z. Dvoi& J. Sci. Food Agric., 32 (1981) 1033. J.W. Dubnoff, in S.P. Colowick and N.O. Kaplan (Eds.), Methods in Enzymology, Vol. 3, Academic, New York, 1957, p. 637. M.M. Barrett, J. Pathol. Bacterial., 42 (1936) 441. P. Eggleton, S.R. Elsden and N. Gough, Biochem. J., 37 (1943) 526. H. Borsook, J. Biol. Chem., 110 (1935) 481. E.P. Diamandis and T.P. Hadjiioannou, Clin. Chem., 27 (1981) 455. E. Mussini, L. Colombo, G. de Ponte and F. Marcucci, J. Chromatogr., 305 (1984) 450. E.R. Ryan and W.H.C. Walker, Microchem. J., 24 (1980) 500. J.F. van Staden, Fresenius’ Z.Anal. Chem., 315 (1983) 141. W.J. Griffiths, Clin. Chim. Acta, 9 (1964) 210.