A modified spectrophotometric procedure for the determination of pyruvic acid in onions using benzaldehyde derivatives.

Abstract

A modified spectrophotometric method for the analysis of pyruvic acid, a compound of agricultural and medical significant is described. The color formation reaction is based on the reaction of pyruvic acid with some benzaldehyde derivatives in an alkaline medium to produce products with yellow colors which exhibit maximum absorbance at 414-446 nm. The monitored spectrophotometric absorbance is evaluated and optimized with respect to various experimental conditions. For the selected benzaldehyde derivative (p-dimethylaminobenzaldehyde), Beer's law is obeyed over the concentration range 5-215 [micro]g [ml.sup.-1] pyruvic acid. The developed analytical procedure shows a good reproducibility, the relative standard deviation RSD% (n=5) at a concentration level of 50 [micro]g [ml.sup.-1] is 0.85% whereas the accuracy of the method is illustrated via the mean recovery of 98.8%. Applicability of the developed method was demonstrated by the determination of pyruvic acid in agricultural samples (onion) and the achieved results were compared with that obtained by reference spectrophotometric method. The statistical analysis (F-test) of these data showed that the results of both analytical methods compared favourably.

Keywords: Pyruvic acid; benzaldehyde derivatives; spectrophotometry; onion.

Introduction

Pyruvic acid is an organic keto acid that has an essential role in biological energy production [1]. Additionally, it is a biologically important molecules involved in a variety of biochemical reactions in plants or animals. From clinical point of view, pyruvic acid has cardiac, skeletal muscle and bariatric activity as well as antioxidant characteristic, hence, it serves as an aid for diagnosing various medical disorders. Pyruvic acid also can promote a multitude of health benefits such as enhancing body weight loss, increasing human body's muscular endurance and boosting exercise performance [2-3]. Fortunately, pyruvic acid is widely found in several agricultural products and the average daily intake of this substance ranges between about 100 milligrams and 1 to 2 grams. Although, certain fruits and vegetables are rich sources for pyruvic acid, however, dietary supplements of it are also commercially available. In the food industry, assaying this compound provides an indication of bacterial contaminations [4]. Moreover, the measurements of the enzymatically produced pyruvic acid is a very reliable indicator of flavour or pungency [5]. Actually, pyruvic acid is formed as a stable primary compound from the enzymatic decomposition of each of the flavor precursors. Measuring pyruvic acid levels as an index of onion pungency has become a routine procedure to ensure the quality of onion for both growers/shippers and consumers [6].

Due to the vital importance of pyruvic acid determination in agricultural and biomedical samples as described above, no wonder that numerous analytical techniques used for its determination have been reported over the years. Such analytical techniques include HPLC [7-9], GLC [10,11], ion chromatography [12], capillary zone electrophoresis [13], chemiluminescence [4,14,15], fluorimetry [16.17], polarography [18], potentiometry [19] amperometry [20,21] amperometric biosensor [22] and fiber optic sensors [23,24]. Spectrophotometric technique is also a very reliable method for such analytical task. Accordingly, a long list of color reagents was accumulated over the years for the determination of pyruvic acid in various real samples. These colour agents include hydroxylamine [25], p-diethylaminocinnamaldehyde [26], methyl indole [27], diazotized 4-chloro-2-nitroaniline [28], 4-nitrophenylhydrazone [29] and Fe[(N[O.sub.3]).sub.3] [30]. However, it is clearly apparent that most of reported spectrophotometric researches in the literature either employed 2,4-dinitrophenylhydrazone (Schwimmer and Weston's procedure) [31] or salicylaldehyde (Straub's procedure) [32] with continues modifications and improvements. Moreover, p-dimethylaminobenzaldehyde (P-DMAB) reagent was also suggested as spectrophotometric reagent for the analysis of pyruvic acid [33,34]. Although p-DMAB color agent was found to be reacted selectively with pyruvic acid and hence it was successfully utilized for its determination, yet the applied method was not simple due to the required reaction conditions for the color development. The originally proposed procedure with p-DMAB demands 60% by volume of dimethylformamide solution as p-DMAB solvent, 25% sodium hydroxide solution and 45 min reaction time at 37[degrees] reaction temperature. Needless to say that such experimental conditions are not ideal from the practical point of view especially in food industry where large number of samples are normally analysed. As a result, the aim of this study is to develop an improved, simple, more reliable and inexpensive spectrophotometric method for the determination of pyruvic acid in some agricultural samples.

Experimental

Apparatus: The spectrophotometric measurements were performed using a Perkin Elmer Lambda 2 spectrophotometer and the required reaction temperatures were achieved via a GFL type ID23 water bath.

Reagent: A stock solution, 1000 [micro]g [ml.sup.-1] of sodium pyruvate (BDH, USA) was prepared by dissolving 1.0 g in 1000 ml distilled water. 0.5% w/v stock solution of p-dimethylaminobenzaldehyde (Aldrich, UK) was prepared by dissolving 0.5g in 100 ml distilled water. Also, 0.2% w/v stock solution of 2,4-dinitrophenylhydrazone (Merck, USA) was prepared by dissolving the appropriate weight in 250 ml of HCL 1 M. 60% w/v stock solutions of sodium hydroxide and potassium hydroxide (BDH, UK) were prepared by dissolving the appropriate weight in distilled water.

Procedure: An aliquot of sodium pyruvate (e.g. 20 [micro]g [ml.sup.-1]) was placed in a 25 ml flask then 2 ml of 0.5% benzaldehyde derivative solution and 2 ml of 60 w/v% KOH solution were added to it. After making up the mixture volume to 10 ml with distilled water, the obtained mixture was heated to the desired temperature with water bath for 15 min for complete color development. The test solution was allowed to cool to room temperature and the spectrophotometric absorption spectra was recorded in the range 350-550 nm against its corresponding blank solution. For the optimization experiments, the absorbance maxima of the resulted colored solution was measured at [[lambda].sub.max] = 414 nm.

Results and Discussion

Absorption spectra

Sodium pyruvate in the presence of p-dimethylaminobenzaldehyde (P-DMAB) solution and potassium hydroxide forms a bright yellowish solution due to the typical aldol condensation reaction given in Scheme 1. The spectrophotometric absorption spectra of the yellow colored product exhibit one band with maximum absorbance at 414 nm as shown in Figure 1. The blank reagent had negligible absorbance at this wavelength. The observed absorbance directly depends on sodium pyruvate concentration and hence can be used for its spectrophotometric determination.

Color development conditions

The chemical and experimental variables had profound affect on the development of the observed color. Thus, its very essential to optimize the reaction conditions.

Selection of benzaldehyde derivatives: In the first approach, the effect of different chemical derivatives was examined. Four representative benzaldehyde compounds, namely, m-hydroxybenzaldehyde (m-HB), p-hydroxybenzaldehyde (P-HB), 2,4-dihydroxybenzaldehude (DHB) and p-dimethylaminobenzaldehyde (P-DMAB) were selected and studied as possible spectrophotometric reagents. According to the absorbance intensity, band shape and width, practical experimental conditions (e.g. reaction temperature, heating time and alkaline concentration) it was found that (p-DMAB) benzaldehyde derivative provided the supreme analytical results, hence, it was used in subsequent studies. Table 1 represents a summary for the practical experimental parameters for all the studied benzaldehyde derivatives.

Effect of KOH concentration: Generally, the applied organic reaction for color development was mainly pH dependent since the monitored analytical signal was only occurs in alkaline medium using potassium hydroxide. Sodium hydroxide base can be also used instead of KOH but with less analytical performance. The effect of varying the KOH concentration on the absorbance intensity over the range 6-30 w/v% is exhibited in Figure 2. The monitored spectroscopic absorbance almost increased linearly with base concentration over the range 6-15 w/v% KOH and decreased thereafter. Accordingly, 15 w/v% KOH concentration was adopted as optimum value. With respect to the used base concentration, the developed spectrophotometric procedure with p-DMAB benzaldehyde derivative was found to be more practically appropriate than the modified Randle reference method, which is a very common used spectrophotometric method for the analysis of pyruvic acid in onions via the utilization of 2,4-dinitrophenylhydrazine (DNPH) color agent [35]. In fact, the Randle reference method required nearly 35 w/v % NaOH concentration.

Effect of p-DMAB concentration: The increase in the concentration of the selected benzaldehyde derivate (p-DMAB) is accompanied by a linear enhancement in the observed spectroscopic signal, which indicates the suitability of the applied approach for the measurement of the studied analyte by calibration graph method. The variation in the absorbance intensity with continuous addition of p-DMAB is illustrated in Figure 3. The linear correlation (r = 0.998) was observed over the range 1 x [10.sup.-3]-12 x [10.sup.-3] w/v% p-DMAB and then start steadily to level off at higher concentration levels. A 12 x [10.sup.-3] w/v% p-DMAB concentration was adequate for further investigations.

Effect of temperature: This experimental parameter greatly enhanced the color formation reaction particularly at low temperature degrees. The effect of heating degree on absorbance intensity was evaluated by subjecting the chemical reaction to a water bath at various temperatures (10-80 [degrees]C) with constant heating time. It was observed that the monitored spectroscopic signal increased dramatically over the range 10-25 [degrees]C and steadily declined afterwards probably due to the decomposition of the formed colored product at high temperatures. The influence of this parameter on the absorbance intensity is shown in Figure 4. Since the room temperature is practically sufficient to ensure the completeness of the color formation, thus, the applied color formation reaction was carried out directly without the need to adjust the reaction temperature at specific value via water baths. Such simple experimental conditions is far more adequate than the Randle reference method which necessitated using 70 [degrees]C heating temperature.

Effect of heating time: Similarly, time of heating is also a vital variable to ensure complete color formation. Therefore, the observed spectrophotometric signal can be further enhanced by optimizing the duration of the heating time. The absorbance intensity study versus heating time demonstrated a sharp enhancement in absorbance intensity over the range 5-15 min (see Fig. 5). However, the recorded analytical signal reached nearly a levelling off stage over the subsequent heating time range (20-60 min). Accordingly, 15 min heating time was considered as optimal value for further studies.

Analytical performance

Before a developed procedure can provide useful analytical information, it is necessary to demonstrate that it is capable of providing acceptable results. Hence, the process of verifying the validation of the proposed spectrophotometric approach and its analytical performance can be carried out by evaluating the following criteria:

Calibration graph and detection limit The calibration curve for the determination of pyruvic acid was prepared according to the analytical procedure under optimum conditions described above and exhibited in Figure 6. A linear relationship between absorbance intensity and pyruvic acid concentration was obtained over a very wide dynamic range and Beer's Law was obeyed over the range from 5 [micro]g [ml.sup.-1] to 215 [micro]g [ml.sup.-1] with 14 data concentration points. Least-squares treatment of the obtained analytical results yielded the following regression equation:

Abs = 0.017 + 0.007 C ([micro]g [ml.sup.-1]) r = 0.9997 n = 14

In addition, the calculated detection limit for the proposed method based on the signal-to-noise ratio (S/N=3) is 3.5 [micro]g [ml.sup.-1] of pyruvic acid (3.9 x [10.sup.-5] mol l-1).

Precision The precision of the applied spectrophotometric method was estimated from the reproducibility of 5 successive measurements of 50 [micro]g [ml.sup.-1] pyruvic acid solution. The precision of the method in terms of the relative standard deviation (RSD) was 0.85 %.

Accuracy The accuracy of this analytical procedure was verified through estimating the recovery of known amount of pyruvic acid (50 [micro]g [ml.sup.-1]). The value of the mean for five recovery tests was calculated to be 98.8%. The accuracy of these analytical results was assessed by the t-test statistical approach. The critical value of t-test at the 95% confidence level is 2.8 and since the calculated t-test value (2.6) is less than the critical value, the null hypotheses is retained. There is no statistical evidence that the mean of the analysed data differ significantly from the known added quantity of pyruvic acid.

Due to the nature of the aldol condensation reaction employed in the in this study, it is possible that some [alpha]-Ketoacid compounds might interfere during the color formation reaction. However, owing to the lack of such interfering reagents in our lab we could not assess their interference affects.

Practical application of the developed method

In order to investigate the applicability of the proposed spectrophotometric method for the measurement of pyruvic acid in agriculture real samples, two different brands of onions were analyzed. To check the validity of the obtained analytical results, the quantity of pyruvic acid in the same onions samples were also analysed via the Randle reference method and the necessary statistical evaluations were carried out. The onion samples with different pungency levels (mild and pungent) were purchased form the local market and their preparation for analysis was carried out according to the method described in the literature [36]. The analytical results obtained are listed in Table 2 and compared with those obtained by the reference method. As can be observed, the content of pyruvic acid varied significantly according to the pungency level of the analyzed onion sample. For instance, five spectrophotometric measurements of pyruvic acid in mild pungency (sweet) onion gave 490 [micro]g [ml.sup.-1] mean concentration level with 27 [micro]g [ml.sup.-1] standard deviation, whereas the pungent (hot) onion gave (as expected) higher pyruvic acid content of 1199 [+ or -] 11 [micro]g [ml.sup.-1]. These results were statistically compared with those obtained by the reported spectrophotometric method (476 [+ or -] 41 [micro]g [ml.sup.-1] and 1195 [+ or -] 14 [micro]g [ml.sup.-1], respectively). The calculated F-test value were 2.31 and 1.62, respectively. The critical value of F-test at the 95% confidence level is 6.39 and since the calculated F-test value did not exceed the theoretical one, there was no significant difference between the developed and reference methods with respect to reproducibility [37].

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Conclusion

As can be extracted from the analytical results shown previously, the proposed spectrophotometric procedure with benzaldehyde derivative is more simple, adequately practical, time-saving and sensitive comparing with the reference spectrophotometric method.

Reference

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Ahmad H. Alghamdi *, Yussef F. Alghtani and S. A. Al-Tamrah Department of Chemistry, College of Science King Saud University P.O. Box 2455, Riyadh--11451, Saudi Arabia 

* Correspondence author E-mail: [e-mail]

Table 1: Summary for practical experimental conditions obtained
for the investigated benzaldehyde derivatives

Benzaldehyde    [lambda]max    KOH Conc.    Derivative
Derivatives                     (w/v%)     Concentration
                                              (w/v%)

m-HB                426 nm          12%           0.135%
p-HB                420 nm          18%           0.201%
DHB                 446 nm          24%           0.075%
p-DMAB              414 nm          16%           0.012%

Benzaldehyde       Temp.       Heating
Derivatives                      Time
                                (min)

m-HB            57 C[degrees]        15
p-HB            45 C[degrees]        30
DHB             75 C[degrees]        64
p-DMAB          25 C[degrees]        15

Table 2: Application of the proposed spectrophotometric method for the
determination of pyruvic acid in onions

      Sweet Onion                    Pungent Onion

Proposed     Reference Method    Proposed    Reference
Method                           Method      Method

   480 *               486       1180          1186
   450                 476       1210          1208
   510                 505       1183          1190
   420                 456       1207          1210
   520                 525       1195          1201
  X: 476               490      X:1195         1199
  s.d: 41               27      s.d:14           11

* All concentrations reported are in [micro]g [ml.sup.-1].