Effects of Fermentation and Storage on Antioxidant Activities of Buffalo and Goat Milk Products

Fermentation of milk enhances its nutritional values through improved bioavailability of nutrients and production of substances including bioactive peptides, which have biological functions.The goals of this research were to study the effect of fermentation and storage on antioxidant activity of buffalo and goat yogurts.Samples of buffalo milk, goat milk and their yogurts were determined for proximate analysis and bioactive activity including antioxidant activities of DPPH, ABTS, reducing power and superoxide assays during the fermentation and their storage. Results showed that buffalo and goat yogurts had antioxidant activities of all assays and their activities significantly increased during their fermentation. However, the activities of both products remained unchange during the storage time of 21 days at 4oC. It can conclude that the antioxidant activity could be found in buffalo and goat yoghurts and the activities were affected by fermentation.The fermentation would lead to protein digestion in milk to produce short chains of amino acids acting as bioactive peptides and lactic acid production possibly responsible for antioxidant activity.

Synergistic effects of recombinant AGAAN antimicrobial peptide with organic acid against foodborne pathogens attached to chicken meat

Background and Objective: Fresh chicken meat includes the capacity to contain foodborne pathogens. A previous study has demonstrated efficacy of recombinant AGAAN antimicrobial peptide against various bacterial strains. In general, AGAAN is a newly discovered antimic-robial peptide with a unique cationic alpha-helical structure. The peptide is originated from the skin secretions of Agalychnis annae. This peptide showed a significant affinity towards the negatively-charged microbial lipid bilayer, as previously demonstrated by the experimental and in-silico analyses. However, the major concerns include high production costs, limited expression, laborious process and potential toxicity associated with concentrated peptides. In this research, the synergistic effects with organic acid were addressed to decrease these problems while preserving its bactericidal activity. Material and Methods: Recombinant AGAAN and organic acids were assessed on Staphylococcus aureus ATCC 6538 and Escherichia coli ATCC 8739. This was carried out by assessing minimum inhibitory concentration and fractional inhibitory concentration. In addition, effects of the combination on bacterial membrane integrity by carrying out beta-gala-ctosidase assessment. Additionally, the potential efficacy of this combination in preserving poultry meat was investigated. Results and Conclusion: Minimum inhibitory concentration of the recombinant AGAAN against the two bacterial strains was 0.15 mg.ml-1. In contrast, the minimum inhibitory concentration of acetic acid against Staphylococcus aureus and Escherichia coli were 0.2 and 0.25% v v-1, respectively. The combination demonstrated significant synergy, as evidenced by fractional inhibitory indices of 0.375 against the two foodborne pathogens. Based on the study, the combination effectively inhibited proliferation of these disease-causing microorganisms that led to foodborne illnesses within 300 min. Presence of intracellular beta-galactosidase indicated that the combination of factors has caused damages to the cell membrane, resulting in its compromised integrity. Red blood cells exposed to various concentrations of recombinant AGAAN and acetic acid did not result in hemolysis. Results showed significant differences (p < 0.05) in all the experiments on meat samples that received treatments with recombinant AGAAN and acetic acid. The current study detected that a combination of recombinant AGAAN antimicrobial peptide with organic acid could effectively inhibit growth of pathogens at lower concentrations. Data presented in this study can help food industries develop further efficient cost-effective antimicrobial uses. Introduction   Prevalence of foodborne diseases has emerged as a significant global health concern. The World Health Organization (WHO) report indicates that nearly 600 million cases of foodborne illnesses occur annually due to the consumption of food substances contaminated with microorganisms and chemicals [1]. Food contamination and increases in the risk of foodborne diseases are caused by pathogenic microorganisms [2]. Meat and meat products are important sources of nutrients for humans due to their high protein composition and other essential nutrients [3].  However, these foods provide appropriate environments for the growth of foodborne microbes due to their high water content and nutrients, [4]. A significant number of studies have shown that Staphylococcus aureus and Escherichia coli are associated with meat contamination [5-7]. The S. aureus is a facultative anaerobic, Gram-positive non-spore-forming bacterium [8]. It is a major problem in foodborne illnesses [9]. The S. aureus infections cause significant morbidity and mortality in developing and developed countries [10]. Similarly, E. coli is a non-spore-forming bacterium and the major cause of foodborne diseases in Gram-negative bacteria. Disease-causing strains of E. coli can infect the stomach, leading to serious abdominal symptoms [11]. Previous studies have primarily concentrated on spore-forming microorganisms, thereby overlooking non-spore-forming ones such as E. coli and S. aureus. Based on their contribution to foodborne illnesses, it is important to develop a cost-effective user-friendly approach to slow their rapid proliferation in food products. Organic acids have been used as antimicrobial agents to inhibit foodborne pathogenic bacterial growth in chicken meats during processing [12]. Due to the potential resistance development by microorganisms, there are needs of drug alternatives that can efficiently kill resistant bacteria and enhance preservation [13]. Antimicrobial peptides (AMP) are produced by living organisms and include critical functions in protecting hosts against infections [14,15]. Likelihood of microbes exhibiting resistance to AMP is exceedingly low because of their wide range of mechanisms of action. Multiple studies have emphasized potential of AMP as a viable option for preventing meat spoilage and foodborne diseases [16-19]. In a previous investigation by the current authors, recombinant AGAAN (rAGAAN) effectively was cloned, expressed and analytically characterized [20]. Technically, AGAAN is a novel antimi-crobial peptide with a cationic α-helical structure from the skin secretions of the blue-sided frogs. The rAGAAN is stable at various temperatures and pH and destroys a wide range of bacteria [20]. A hemolytic assay has shown that the peptide is relatively non-toxic to mammalian red blood cells (RBCs). Combination of these characteristics with its rapid killing kinetics demonstrates that rAGAAN includes the potential as an effective food preservative against foodborne pathogens. Nevertheless, major issues include exorbitant production expenses, labor-intensive procedures and potential toxicity of using high concentrations of peptides. Combining two or more AMPs may boost antimicrobial activity at lower doses [21]. The present study assessed pairwise combinations of the rAGAAN with formic and acetic acids against E. coli and S. aureus. Selection of these two organic acids was based on their high effectiveness against the highlighted bacterial strains. In addition, FAO/WHO Expert Committee on Food Additives has classified acetic and formic acids as generally regarded as safe. The former chemical was assigned to an unrestricted group acceptance daily intake (ADI), while the latter was assigned to an ADI range of 0–3 mg.kg-1 [22]. Combination of rAGAAN and these organic acids could decrease the concentration while preserving their potentially bactericidal activity. Differences in their mechanisms of action necess-itate assessment of synergy in membrane permeation and kinetics of inactivation. This study could provide an additional option for poultry industries to protect chicken meats from pathogens. Materials and Methods 2.1 Bacterial strains Department of Microbiology at King Mongkut's University of Technology Thonburi in Bangkok, Thailand, supplied the foodborne pathogenic strains of E. coli ATCC 8739 and S. aureus ATCC 6538. 2.2 Recombinant AGAAN peptide expression and purification The rAGAAN was produced based on the method of Ajingi et al., [20]. Briefly, the recombinant plasmid (pET-AGAAN) was transformed into E. coli BL21 (DE 3) competent cells. A colony of the competent cells with recombinant plasmids was inoculated into Luria-Bertani (LB) broth supplemented with chloramphenicol and ampicillin and grown at 37 °C and 200 rpm overnight. Then, 1% v v-1 from the overnight culture was introduced into a fresh 1-l LB broth supplemented with chloramphenicol and ampicillin as well as 1% w v-1 glucose. Culture was grown to an optical density (OD 600 nm) range of 0.4–0.6 at 37 °C and 200 rpm. Then, rAGAAN was expressed through induction with isopropyl β-D-1-thiogalactopyranoside at a concentration of 500 mM. Culture was grown at 16 °C for 18 h at 150 rpm. Cells were collected through centrifugation at 6,120× g for 30 min at 4 °C. Then, cells were suspended in 10 ml of buffer solution (10 mM Tris-HCl, 1 M NaCl; pH 8.0). These were subjected to sonication at an amplitude of 60% for 2 min, repeated for five cycles to induce cell disruption. Supernatant was purified after sonication and centrifugation at 6,120× g for 25 min at 4 ℃ using HisTrap FF column linked to the FPLC system. The column was pre-equilibrated with binding buffer (10 mM Tris-HCl, 1 M NaCl; pH 8.0). Elution of the bound peptide was carried out using buffer B (10 mM Tris-HCl, 1 M NaCl, 250 mM imid-azole; pH 8.0). Then, dialysis was carried out overnight at 4 °C using 50 mM Tris-HCl solution. Then, peptide was concentrated using 3-kDa centricon centrifugal filter tubes (Amicon, Germany). Concentration of the rAGAAN was measured using Bradford protein assay and its purity was assessed using 16% tricine-sodium dodecyl sulfate–polyacrylamide gel electrophoresis (tricine-SDS-PAGE). 2.3 rAGAAN and organic acid preparation The rAGAAN was formulated in milligrams per milliliter (mg.ml-1). It was dissolved in 1× phosphate-buffered saline (PBS), whereas the organic acids were formulated in percentages (% v v-1) by dissolving in distilled water (DW). 2.4 Culture preparation A volume of 20 μl of microbial stock, previously stored at -80 ◦C, were plated on LB agar. The resulting culture was incubated at 37 oC for 18 h. Then, subculture process was carried out for each strain under identical conditions to preserve integrity and purity of the cells. On the next day, a suspension was generated by transferring isolated colonies into sterilized 10-ml LB media. The bacterial strains were cultured until they reached an OD of 108 cfu.ml-1. This measurement was achieved at 600 nm using spectrophot-ometer (U-2900UV/VIS Hitachi Tokyo, Japan). Concen-tration was modified to 105 cfu.ml-1 using sterile LB broth. 2.5 Minimum inhibitory concentration assessment Briefly, 50 μl of the inoculated sample were administered into each well of the 96-well plates. Then, aliquots of 50 μl were dispensed into the wells, containing rAGAAN and organic acids at various concentrations. The 96-well plates with the lids closed were incubated at 37 °C for 18 h. Results were analyzed at 600 nm using microplate reader (BioTek, synergy H1, Winooski, USA). Control contained 100 μl of the bacterial inoculum. The MIC values included the lowest concentrations of the antimicrobial agents that cause bacterial growth inhibition. 2.6 Synergistic effects of rAGAAN with acetic and formic acids Combination effects of rAGAAN with organic acids against the bacterial strains were assessed using checkerboard method. Briefly, 18-h cultures in LB broth were used to inoculate fresh LB broth to achieve a cell density of approximately 105 cfu.ml-1. Generally, 50 μl of the inoculated sample were added into 96-well microplates. Then, rAGAAN and organic acids were transferred into the 96-well microplates with increasing concentrations arran-ged in columns and rows, respectively. The organic acids were mixed with rAGAAN separately to assess their combinatorial effects on pathogenic bacteria. The purpose was to decrease the effective concentration of rAGAAN while preserving its antimicrobial activity. Assessment of the synergistic interactions involved the summation of the fractional inhibitory concentration indices (FICI) as Eq. 1 [23].                                                                                                  Eq. 1 where, FICI ≤ 0.5 indicated synergistic relationships between the rAGAAN and organic acids that increased the antimicrobial activity, FICI > 0.5–4.0 was indifferent and FICI > 4.0 was antagonistic. 2.7 Kinetics of inactivation The OD of bacterial strain was measured to assess the rate of inactivation when treated with rAGAAN, acetic acids or their combination. Bacterial culture, diluted in LB broth to a concentration of approximately 105 cfu.ml-1, was added to 96 well plates. The rAGAAN and acetic acid were added at their minimum inhibitory concentration (MIC) levels, individually and in combination with their fractional inhibitory concentration (FICI) at 1×, 2× and 3× to separate wells. The 96-well plate was incubated at 37 °C. The procedure entailed monitoring the rate of inactivation for various bacterial strains by measuring the OD at consistent intervals of 1 h for 5 h. The OD was measured using spectrophotometer set at 600 nm and microplate reader (BioTek, synergy H1, Winooski, USA). 2.8 β-Galactosidase assay The β-galactosidase assay was carried out to assess effects of the rAGAAN and acetic acid or their combination on membranes of the bacteria. First, E. coli was inoculated into lactose broth and incubated at 37 °C for 18 h to stimulate β-galactosidase production. The bacterial cells were centrifuged and the pellet was washed thrice with 1× PBS. Then, the bacterial concentration was modified to roughly 105 cfu.ml-1 in 1× PBS solution. Moreover, 50 μl of purified rAGAAN, acetic acid and their combination at 1× FICI, 2× FICI and 3× FICI were added into wells of a microplate containing 50 μl of E. coli cell suspension. A volume of 30 μl of O-nitrophenyl-β-D-galactoside (ONPG) were added into every well of the microplate. The microplate was incubated at 37 °C and activity was assessed by measuring the spectrophotometric absorbance at 405 nm and various time intervals. 2.9 Hemolysis assay The RBC lytic assay was carried out based on a procedure by Taniguchi et al. [24] with minor adjustments. The RBCs were washed thrice in 1× PBS and centrifuged at 14,530× g for 10 min. Pellet was dissolved in 1× PBS to achieve a concentration of 4%. Generally, 500 μl of blood were mixed with 500 μl of rAGAAN and acetic acid, indivi-dually and in various combinations (1×FICI, 2×FICI, and 3×FICI). The positive control included a solution containing 0.1% TritonX-100, while the negative control included a solution containing 1× PBS. Solution was incubited in microtubes at 37°C for 1 h and centrifugation was carried out at 14,530× g for 5 min. Then, 100 μl of the supernatant were extracted from each microtube and transferred to each well of 96-well plate. Assessment of hemoglobin release was carried out by measuring the absorbance at 540 nm. 2.10 rAGAAN-acetic acid against chicken meat spoilage Antimicrobial efficacy of the rAGAAN and acetic acid combination was assessed using a methodology described by Ajingi et al. [25], with minor adjustments. In brief, fresh chicken meat was purchased from a local market and immediately transferred to the laboratory. Meat was divided into approximately 10-g specimens and washed thoroughly. Specimens were transferred into a laminar flow hood and 100 µl of 105 cfu of E. coli were divided to five separate locations. Sample was set for 1 h to promote appropriate attachment of the bacterial strains. Then, meat sample was submerged into 200-ml solution of rAGAAN/acetic acid for 1 h. Furthermore, sample was extracted, transferred into a plastic bag and incubated at 37 oC for 3 d. The chicken meat sample was transferred into a plastic bag with solution consisting of 0.1% peptone water. Sample was mechanically pulverized using stomacher to enhance liberation of the bacterial cells. Following the process of serial dilution, a 100 µl of sample were transferred onto an LB-agar plate. Number of colonies on the plate was counted at intervals of 0, 1, 2 and 3 d. Control group was administered with DW. 2.11 Statistical analysis Results were present as mean ±SD (standard deviation) of three replicates. Statistical distinction was assessed using one-way analysis of variance (ANOVA) with Duncan’s multiple-range test. Differences with p < 0.05 were regarded as statistically significant. Results and Discussion 3.1 Minimum inhibitory concentration The MICs of rAGAAN and organic acids against S. aureus and E. coli are present in Table 1. The MIC of rAGAAN against S. aureus and E. coli was assessed as 0.15 mg.ml-1. The organic acids inhibited proliferation of the pathogenic bacteria at various concentrations expressed as proportions (%). Acetic acid demonstrated inhibitory effects on the growth of S. aureus at 0.2% v v-1 and on the growth of pathogenic E. coli at 0.25% v v-1. Formic acid inhibited growth of S. aureus at 0.25% v v-1 and growth of E. coli at 0.2% v v-1. The findings for acetic acid were similar to those against eleven mastitis pathogens in dairy cows with MIC values ranging of 0.125–0.25% v v-1 [26]. Similarly, Fraise et al. [27] reported antimicrobial activity of acetic acid against Pseudomonas aeruginosa and S. aureus at 0.166 and 0.312% v v-1, respectively. Manuel et al. [28] detected that formic acid at a concentration of 0.06% v v-1 exhibited antimicrobial effects against E. coli. Variations in their effectiveness against the microorganisms might be attributed to their chemical compositions. Methyl group (CH3) in acetic acid donated electron density to O-H bond, resulting in increased difficulties in removing the hydrogen atom. Consequently, acetic acid was weaker than the formic acid. Weak acids included a higher ability to pass through bacterial membranes, compared to strong acids due to the balances between their ionized and non-ionized states. The non-ionized form could easily diffuse through hydrophobic membranes. As a result, they provided proton gradients needed for ATP synthesis to collapse. This occurred because free anions such as acetate in this situation combined with periplasmic protons that were pumped out by the electron transport chain. Then, anions transported the protons back across the membrane without F1Fo ATP synthase [29]. 3.2 Synergistic effects of rAGAAN with organic acids The inhibitory concentration index (FICI), demons-trating combined effects of rAGAAN and organic acids, is present in Table 2. The compound rAGAAN demonstrated synergistic effects against S. aureus and E. coli when combined with organic acids. Results showed that the synergistic effects were strongest when using acetic acid for the two bacterial strains, compared to when using formic acid. The FICI values for the combination of rAGAAN with acetic acid were assessed as 0.375 (p < 0.5) for S. aureus and E. coli. The FICI values for the combination of rAGAAN with formic acid were assessed as 0.375 for S. aureus and 0.5 for E. coli.         Results indicated that sub-MICs of the antimicrobials were needed to effectively terminate the bacterial growth. Combination of rAGAAN and acetic acid resulted in a 25% decrease in the concentration of each antimicrobial, compared to their MICs. Synergism can occur when two various antibacterial agents, each with non-overlapping mechanisms of action, are combined with each other [30]. Therefore, the authors suggest that the antimicrobial effects could be strengthened using synergistic effects of combined organic acid with rAGAAN. While the precise process; by which, combination of rAGAAN with organic acid created synergistic effects is still unknown, studies have demonstrated that the cell membrane of bacteria is a shared target for the antibacterial effects of various antimicrobial peptides. Additionally, these peptides include an affinity for bacterial cellular components, including DNA [31]. In contrast, it is suggested that organic acids can delay absorption of nutrients and disrupt flow of electrons, leading to decreases in ATP production [32]. This various mechanism of action enables swift eradication of bacteria. 3.3 Kinetics of inactivation Acetic acid was chosen for the study because it included stronger antimicrobial effects than that formic acid with rAGAAN did. Growth inhibition kinetics of rAGAAN, acetic acid and their combination on the logarithmic phase of the pathogenic bacteria are illustrated in Figure 1. When the peptide rAGAAN was mixed with acetic acid at the FIC, there was no noticeable alteration in OD for either of the bacterial strains during 5 h. This indicated that the bacterial growth was entirely suppressed. The combination demonstrated significant inhibitory effects, greater than that of the individual antimicrobial agent and control group. The combination exhibited the capacity to inhibit proliferation of S. aureus ATCC 6538 and E. coli ATCC 8739 at various concentrations within a few hours of exposure. Upon analyzing each treatment individually, it became clear that progressive decreases occured in OD measurements as time progressed. Nevertheless, use of rAGAAN with acetic acid led to further pronounced decreases in the turbidity level of the culture. 3.4 β-Galactosidase assay To clarify the mechanism; by which, the combination acted, membrane permeability assay was carried out. This experiment used E. coli that was cultured in media containing lactose broth, which stimulated the synthesis of β-galactosidase. The β-galactosidase is an endogenous enzyme synthesized by the lac operon in bacteria. Release of this enzyme depends on disruption of the cell membrane. Release of the β-galactosidase enzyme from the disrupted cytoplasmic membrane was detected within 10 min of incubation with rAGAAN alone. In addition, cell membrane was destabilized by a combination of rAGAAN with acetic acid at 1× FICI, 2× FICI and 3× FICI, as shown in Figure 2. Findings showed that the presence of rAGAAN, independently and in combination with acetic acid, could result in permeability of the cell membrane of E. coli. How-ever, acetic acid alone did not demonstrate effects on perme-ability of the membrane. Increasing OD measurements over time were directly linked to the rate of O-nitrophenol prod-uction from the breakdown of ONPG. The current results were similar to those of Yuan et al. [33], who observed increases in OD due to the degradation of ONPG when Larimichthys crocea myosin heavy chain protein-derived pepti

Toxic Compound, Anti-Nutritional Factors and Functional Properties of Protein Isolated from Detoxified Jatropha curcas Seed Cake

Jatropha curcas is a multipurpose tree, which has potential as an alternative source for biodiesel. All of its parts can also be used for human food, animal feed, fertilizer, fuel and traditional medicine. J. curcas seed cake is a low-value by-product obtained from biodiesel production. The seed cake, however, has a high amount of protein, with the presence of a main toxic compound: phorbol esters as well as anti-nutritional factors: trypsin inhibitors, phytic acid, lectin and saponin. The objective of this work was to detoxify J. curcas seed cake and study the toxin, anti-nutritional factors and also functional properties of the protein isolated from the detoxified seed cake. The yield of protein isolate was approximately 70.9%. The protein isolate was obtained without a detectable level of phorbol esters. The solubility of the protein isolate was maximal at pH 12.0 and minimal at pH 4.0. The water and oil binding capacities of the protein isolate were 1.76 g water/g protein and 1.07 mL oil/g protein, respectively. The foam capacity and stability, including emulsion activity and stability of protein isolate, had higher values in a range of basic pHs, while foam and emulsion stabilities decreased with increasing time. The results suggest that the detoxified J. curcas seed cake has potential to be exploited as a novel source of functional protein for food applications

Physical and chemical properties of fish and chicken bones as calcium source for mineral supplements

Physical and chemical properties of two bones of two species of fish, hoki (Macruronus novaezelandiae) and giant seaperch (Lates calcarifer Bloch.), were compared with chicken bone to evaluate their composition for use as natural calcium supplement. The information could be useful for waste utilization in the food and pharmaceutical industries. Physical testing and chemical analyses were performed according to the USP 24 and BP 1998 standards under calcium carbonate monograph. Loss on drying found in hoki, giant seaperch and chicken bones was 12.4, 11.3 and 5.9 % w/w, calculated on dried basis, respectively. Total calcium determined by complexometric titration was 31.8, 28.1 and 32.2% w/w in hoki, giant seaperch and chicken bones, respectively. All samples contained carbonate and phosphate anion residues but gluconate, acetate and citrate were absent. The presence of calcium carbonate was confirmed by thermogravimetry. Results from all bones showed that limit tests for heavy metals, arsenic and iron complied with the USP standard, whereas barium, chloride and sulfate conformed to the BP standard. The magnesium and alkali metals in giant seaperch bone were within the BP limit (1.5%), but those of hoki and chicken bone exceeded the limit

Bacterial Selection from Shrimp Ponds for Degradation of Organic Matters

Accumulation of ammonia, nitrite and hydrogen sulfide in a shrimp pond is generally caused by incomplete degradation of residual organic matters from overfeeding and from organic wastes released by shrimps. The phenomenon affects shrimp growth and survival rate. The objectives of this investigation were to screen for a bacterial strain able to digest organic residues and to evaluate the changes of residues by bacterial activities under natural conditions. The results from this work showed that the isolated strain, Bacillus cereus S1, had the highest protease activity (57.1 U/ml) with the presence of glucoamylase and lipase activities (4.5 and 0.13 U/ml, respectively). Under an aseptic condition in 1-L flasks containing seawater with 0.1% shrimp feed, B. cereus S1 degraded organic matters and significantly reduced chemical oxygen demand (COD) (70.8%). An amount of ammonia-nitrogen was increased during the first 5 days of incubation due to the degradation of organic compounds in shrimp feed. However, it declined afterward with nitrate-nitrogen increase and unchanged nitrite nitrogen content. Under natural conditions in 10-L glass jars containing seawater with 0.05% shrimp feed and 0.05% sediment, B. cereus S1 and a commercial bacterial product (Inpicin-G) could reduce COD (4.5% and 15.8%, respectively) and biochemical oxygen demand (BOD) (35.1 and 11.4%, respectively). However, similar changes of ammonia-nitrogen, nitrate-nitrogen and nitrite-nitrogen contents in water samples were observed. The results indicate that this selected bacterium could reduce organic compound accumulations on a laboratory scale. In addition, the strain did not produce any enterotoxins compared to a toxin standard. Therefore, the bacterium, Bacillus cereus S1, could be applied to decrease organic matters accumulated in shrimp pond without any harm to shrimps or consumers

Spectrophotometric method for the simultaneous determination of piroxicam and 2-aminopyridine

737-740A rapid and simple spectrophotometric method is proposed for the simultaneous determination of piroxicam (Pi) and 2-aminopyridine (2-Ap). Piroxicam is stable under basic hydrolysis, but yields 2-Ap as one of the degradation products under acid hydrolysis. The method is based on the measurement of absorbances of 2-Ap and Pi at 300 and 360 nm, respectively, and the calculations are based on the binary method. The absorbances of both compounds obey Beer-Lambert 's law over the concentration range of 5-25 µg L-1 with good linearity (r2>0.99). The recoveries are with in 100.8- 106.4% for Pi and are within 96.4-98.9% for 2- Ap. Precision is good with acceptable limits of detection (LOD) and quantitation (LOQ) for both compounds. The method has been applied for the determination of Pi and 2-Ap in piroxicam capsules. The average content of two different brands of piroxicam is 97.4 and 98.5% (n = 3), which complies with the USP 26 (92.5- 107.5%). Under the stress condition (refluxing with 0.1 N HCI), the percentages of piroxicam decrease from 100% (0 h) to 18.9% (21 h) and 2-Ap increase from 0% (0 h) to 63.6% (21 h)

Method Development for Separation of Active Ingredients in Cold Medicines by Micellar Electrokinetic Chromatography

Separation of nine commonly used active ingredients in cold medicines, were demonstrated by micellar electrokinetic chromatography. The ingredients included paracetamol, chlorpheniramine maleate, diphenhydramine hydrochloride, triprolidine hydrochloride, phenylpropanolamine hydrochloride, dextromethorphan hydrobromide, loratadine, aspirin and caffeine. Effects of buffer concentrations, pH, organic modifiers and capillary length were investigated. The optimum conditions were achieved in 10 mM sodium dihydrogenphosphate-sodium tetraborate buffer, pH 9.0, containing 50 mM sodium dodecyl sulfate and 28% v/v acetonitrile using the effective length of 50 cm, the separating voltage of +15 kV and the capillary temperature of 30°C. Separation of all peaks was obtained within 28.4 min with a resolution of 1.2