Journal of Life Sciences Volume 6 Number (2)

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Journal of Life Sciences

Volume 6, Number 7, July 2012 (Serial Number 51)

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Journal of Life Sciences

Volume 6, Number 7, July 2012 (Serial Number 51)

Biochemistry and Medical Science

713 Lipid Oxidation and Histamine Production in Atlantic Mackerel (Scomber scombrus) Versus Time

and Mode of Conservation

Hanane Oucif, Smail Ali-Mehidi and Sidi-Mohamed El-Amine Abi-Ayad

721 In Vitro Regeneration of Commercial Sugarcane (Saccharum spp.) Cultivars in Nigeria

Sani Lawan Abdu, Mustapha Yahaya and Usman Inuwa Shehu

726 Validation of Analytical Method of Irbesartan Plasma in Vitro by High Performance Liquid

Chromatography-Fluorescence

Harmita, Yahdiana Harahap and I. Kadek Arya M.

732 Isolation, Phenotypic Identification and Fingerprint by RAPD-PCR of Oxalotrophoic

Burkholderia cepacia from Egyptian Fertile Soil

Hala Abou Shady, Mona Kilany Abd El Gawad, Qadria Genedi and Wafaa Farouk

740 The Effect of a Dietary Supplement Spirulina and Bifidobacterium adolescentis on the

Cholesterol-Lowering in Vitro and in Vivo

Amel Doumandji, Dahmane Alili and Abderrahmen Benzaiche

747 Attitudes and Practices of Health Care Providers Regarding the Management of Uncomplicated

Malaria in Abidjan, Cote d’Ivoire

Frederic Nogbou Ello, Thomas Yapo Aba, Ismael Ouattara, Didier Koumavi Ekouévi, Offoue Kra, Pierre Aimé Assemian, Amath Wade, Serge-Paul Eholie and Emmanuel Bissagnene

754 On the Reproduction Number and a Presentation of Results for Infectious Diseases Models

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Botany and Zoology

758 High Temperature and Abscisic Acid Modified the Profile of Anthocyanins in Grape (Vitis

vinifera L.)

Leonor Deis, Maria Inés de Rosas and Juan Bruno Cavagnaro

766 Substrate Type Affects Growth, Yield and Mineral Composition of Cucumber and Zucchini Squash

Mariateresa Cardarelli, Youssef Rouphael, Salem Darwich, Elvira Rea, Antonio Fiorillo and Giuseppe Colla

771 Effect of Compost Based Substrate and Mycorrhizal Inoculum in Potted Geranium Plants

Monica Tullio, Federico Calviello and Elvira Rea

776 The Breeding Status of the Glossy Ibis Plegadis falcinellus in the Lebna Dam in Cap Bon, Tunisia

Aymen Nefla, Ridha Ouni and Saïd Nouira

783 Survey on Parasites in Sparrows of Amol (Mazandaran Province, Iran)

Sina Faghihzadeh Gorji, Bahareh Shemshadi, Hamid Habibi, Sattar Jalali, Masoud Davary and Mohammadreza Sepehri

Interdisciplinary Researches

786 Incidence and Development of Powdery Mildew (Blumeria graminis f.sp. tritici) in the Czech

Republic in the Years 1999-2010 and Race Spectrum of This Population

Lubomir Vechet

794 Poverty and Agroforestry Adoption: The Cases of Mucuna pruriens and Acacia auriculiformis in

Godohou Village (Southern Benin)

Emile N. Houngbo, Anne Floquet and Brice Sinsin

801 Modelling Stand Dynamics after Selective Logging: Implications for REDD and Carbon Pools

Estimations from Forest Degradation

Adrien Njepang Djomo, Gode Gravenhorst, Anthony Kimaro and Marney Isaac

817 Aqua Forestry and Duck Integration in Tamil Nadu, India

Faroque Rahamathulla Sheriff

826 The Importance of Fiber Production for Conservation of Native Sheep and Goat Breeds and

Silkworm Lines in Turkey

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Journal of Life Sciences 6 (2012) 713-720

Lipid Oxidation and Histamine Production in Atlantic

Mackerel (

Scomber scombrus

) Versus Time and

Mode of Conservation

Hanane Oucif, Smail Ali-Mehidi and Sidi-Mohamed El-Amine Abi-Ayad

Lab. of Aquaculture and Bioremediation, Dpt. of Biotechnology, University of Es-Sénia, Oran 31000, Algeria

Received: December 11, 2011 / Accepted: February 15, 2012 / Published: July 30, 2012.

Abstract: Lipids oxidation, histamine production and quality loss were studied according to storage time and temperature (ambient temperature (Tamb) 26 °C, 4 °C and –18 °C) in Atlantic mackerel (Scomber scombrus). Muscle pH, hydrolysis of phospholipids,

content of primary (hydroperoxides), secondary lipid oxidation products (TBARS) and histamine were determined and compared with a sensory assessment. Atlantic mackerel is sensory acceptable, less than 24 hours at Tamb, for up to 3 days at 4 °C and 3 months

at –18 °C. Evolution of biochemical parameters with storage time and temperature showed significant differences (P < 0.05). Muscle pH increased from 5.99 to 6.13 at Tamb, to 6.23 at 4 °C and to 6.04 at –18 °C. The highest content of TBARS is associated with a

decrease in phospholipids and hydroperoxides contents and highest levels of sensory alteration. Histamine content exceeded the limit recommended by the Trade Algerian Ministry (10 mg/100g), after 24 hours at Tamb, 5 days of storage at 4 °C only. Therefore, freezing

storage has a preserving effect on lipid damage and histamine production and seems the best means of storage; if these species are not consumed during the two days following capture. Moreover, monitoring histamine production is more useful as sanitary index rather than spoilage parameter and the strategy used for measuring kinetic of lipid oxidation appear pertinent for determining the degree of oxidation.

Key words: Conservation, histamine, hydroperoxides, phospholipids, temperature, TBARS, Scomber scombrus.

1. Introduction



Seafood has attracted considerable attention as a source of high amounts of important nutritional components to human diet such as proteins of high biological quality; it is also exceptionally rich in polyunsaturated long chain fatty acids, in particular the omega 3 (PUFAs-ω3) and minerals [1].

However, it is well known to deteriorate rapidly, especially in hot countries which lack adequate infrastructure of conservation. Indeed, immediately after his capture, fish undergoes a natural process of decomposition as a result of successive enzymatic and chemical reactions and bacterial infections. Hydrolysis and lipid oxidation in particular, is certainly one of the

Corresponding author: Hanane Oucif, Ph.D., research field: quality control. E-mail: [email protected].

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Lipid Oxidation and Histamine Production in Atlantic Mackerel (Scomber scombrus) Versus Time andMode of Conservation

714

process can destroy [3]. These two process changes represent a real danger for consumer. Therefore it is necessary to impose development of modern and traditional methods to ensure the quality of food.

The present experiment was carried out on Atlantic mackerel (Scomber scombrus), one of abundant pelagic fatty fish species, from Scombridae family, often implicated in cases of histamine poisoning and in the present work, biochemical and sensory changes that take place during storage of Atlantic mackerel are investigated by evaluated lipid damage and histamine value during storage period at –18 °C, 4 °C and ambient temperature and complemented by sensory analysis.

The objective is to identify the critical points of different treatments of preservation, at low temperatures (4 °C and –18 °C) and ambient temperature (26 ± 3 °C) and to develop guidelines and index that reflect freshness and can be measured, using simple and rapid methods.

2. Materials and Methods

2.1 Raw Material, Sampling and Processing

Fresh Atlantic mackerel (Scomber scombrus, 140.51 ± 14.10 g) are bought from fishing port of Oran and kept on ice till arrival to the laboratory (6 hours). Part of the fish is directly packaged in polyethylene bags and immediately frozen at –18 °C, and the other is divided into two batches, one is stored at ambient temperature (26 ± 3 °C, and at 4 °C at night) and the second one is stored at 4 °C (24 h/24 h). Samples are taken for analysis at day 1, 2, 3, 5, 7 and 9 from storage at 4 °C and ambient temperature and at day 15, 30, 60, 90 and 120 from storage at –18 °C. In each batch at both temperatures, analyses are carried out on five mackerels.

2.2 Sensory Analysis

Sensory analysis is conducted according to the traditional guidelines [4]. Four categories are ranked: highest quality (E), good quality (A), fair quality (B)

and unacceptable quality (C). This sensory assessment evaluate freshness by analyse certain aspect of general appearances (skin, external odour, gills, consistency and flesh odour).

2.3 Chemical Composition

Evolution of pH values in fish muscle along the storage time is carried out according to the method described by Özogul et al. [5]. The lipid fraction is extracted from 5 g of sample after homogenisation in chloroform/methanol (2:1, v/v), according to the Folch et al. [6] method. Then phospholipids content is measured by colorimetric method of Stewart [7] based on formation of a complex between phospholipids and ammonium ferrothiocyanate. A standard curve is made with standard phosphatidylcholine in chloroform and results are expressed as g phosphatidylcholine equivalents per kg of wet weight (g Eq. PC/kg W.W.).

2.4 Lipid Damage Measurements

Lipid hydroperoxides content expressed as mmoles of cumene hydropeoxide equivalents (CuOOH Eq) per kg of wet weight is determined according to the method of Eymard & Genot [8]. The secondary compounds of lipid oxidation (TBARS) are determined by a method of Genot [9]. Results are expressed as mg of malonaldehyde equivalents per kg of wet weight.

2.5 Histamine Analysis

Histamine quantification in fish muscle is carried out by a colorimetric method of Patange et al. [10], based on the interaction between the imidazole ring and p-phenyldiazonium sulfonate. Concentration of histamine in sample is obtained from the standard curve and expressed as mg per 100g of wet weight (mg/100g W.W.).

2.6 Statistical Analysis

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variance) and by non-parametric variance analysis of Kruskal-Wallis and Mann-Withney U test (for non homogeneous variance), after verification of variance homogeneity by Hartley test.

3. Results and Discussion

3.1 Sensory Assessment

At ambient temperature (26 °C)/chilled (4 °C), sensory evaluation of mackerel indicates that the keeping time is less than 24 hours. Comparable times have been reported within 24 hours [11], 7 hours at 30 °C [12] and 23 hours at 21-27 °C [13] in sardine and 12 hours at 20-25 °C, with anchovies [14]. Upon storage, fish has a rancid smell increasingly strong; as a result of biochemical changes such as degradation of ATP [15] and formation of propanol, pentanol and ethanol; products of lipid peroxidation [16, 17]. These rancid odors become sulfuric smells, following the growth of microorganisms producing H2S particularly at ambient temperature [18]. In addition, Cheuk et al. [19] report that there is a strong correlation between productions of volatile nitrogenous bases, responsible for unpleasant odors and bacterial growth. Advanced decay in some cases is due to the relative content of lipid species (fatty fish deteriorates quickly) [20]. During storage at 4 °C, mackerel is considered unfit for consumption beyond three days. These delays are consistent with other results, including 3 days in sardine [21], 4 days in Trachurus mediterraneus [22]. Delays a little longer were reported: 5 or 6 days in T.

trachurus [23, 24], 5 days [11, 25] in sardine and 4-6

days in anchovy [14]. These relatively short periods of acceptability can be explained by the predominance of psychrotrophic bacteria and gram-negative psychrophilic in fish caught in cold and temperate waters [26]. These differences in shelf life can be attributed to fishing areas that influence the character of psychrophilic or mesophilic natural flora of fish [16]. By way of freezing (–18 °C), the samples are very good for 2 months and unacceptable beyond 3 months (Table 1). A preservative period of 5 months

Table 1 Comparative sensory acceptability of mackerel’s lots.

Storage Days

1 2 3 5 7 9 15 30 60 90 120

At 26 °C/4°C E C C

At 4 °C E A B C C C

At –18 °C E E E A B C

E: highest quality; A: good quality; B: fair quality; C: unacceptable quality.

was proposed in mackerel stored at –20 °C, preceded by freezing at –80 °C for 24 hours [27].

3.2 The pH Evolution

Initial pH value of muscle is 5.99. In mackerel, red muscle is well developed and contains more glycogen than white muscle [28]. It appears that the breakdown of glycogen to lactic acid makes pH of fish after capture, acid, especially in mackerel [29]. Evolution of pH is dependent on mode of storage (P < 0.05) (Fig. 1). As a result, pH values are lower at low temperatures. The increase in pH is due to production of volatile bases (NH3, TMA and DMA) and an accumulation of peptides and amines [30]. It has been reported in sardine [31] and mackerel [16]. However, the decrease in pH is recorded in early days of storage under refrigeration (4 °C). This is not observed at ambient temperature because bacterial multiplication is rapid with a short or no lag phase [13]. The small difference in pH in terms of absolute values, from the beginning

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to the end of different modes of preservation; incentives Laghmari & El Marrakchi [32] to conclude that pH cannot be considered as reliable for assessment of spoiled fish.

3.3 Lipid Damage Analysis

Initially, phospholipids content (PL) is 260.72 g Eq. PC/kg W.W.). Hydrolysis of PL is the main factor of lipids degradation [17, 33] and particularly for mackerel stored at ambient temperature/chilled and refrigerated at 4 °C. Indeed, at ambient temperature, we are witnessing a breakdown of weak bonds [34]. However, in mackerel, phospholipases seem inactive from the seventh day at 4 °C (Fig. 2). Indeed, it appears that the activity of phospholipases decreases after 7 days of storage of mackerel at 2-3 °C [35], which probably the explanation of a sudden increase in the amount of PL. However, she found the initial PL value at Day 9; this suggests that it is likely due to interindividual variations within the species [36] or possibly an accumulation of phospholipids after total inactivation of phospholipases. Moreover, there is any significatively difference between storage at 26 °C and at 4 °C (P≥ 0.05) During freezing at –18 °C, the decrease (P < 0.05) then stability (from 2 months) of the PL content of mackerel are likely due to excessive lowering of temperature, which has the effect of slowing down and braking hydrolysis of PL and in

Fig. 2 Temporal evolution of phospholipids content (g Eq. PC/kg W.W.), of Atlantic mackerel (S. scombrus) of lots A, B and C. Histogram’s (same storage conditions) with different indices are significantly different (P < 0.05).

the same way so; phospholipases. Lipolytic activity, although small, continues during storage in the frozen state [37] and can become the limiting factor for duration of freezing, this is especially true for fatty fish [38].

Hydroperoxides record a maximum on day 2 (propagation phase) (P < 0.05) at ambient temperature and 4 °C, respectively. During the first three days, TBARS (Thiobarbituric acid reactive substances) are relatively low and stable; this observation was also reported by Simeonidou et al. [24], during ice storage of fatty fish. Followed by, a decrease of hydroperoxides, due to their interactions with biological components or their decomposition [39]. Indeed, after 5 days, spread of TBARS is launched (P

< 0.05) (Figs. 3, 4). These secondary products of lipid

Fig. 3 Temporal evolution of hydroperoxides content (mmol Eq. CuOOH/kg W.W.), of Atlantic mackerel (S. scombrus) of lots A, B and C. Histogram’s (same storage conditions) with different indices are significantly different (P < 0.05).

Fig. 4 Temporal evolution of TBARS content (mg Eq. MDA/kg W.W.), of Atlantic mackerel (S. scombrus) of lots A, B and C. Histogram’s (same storage conditions) with different indices are significantly different (P < 0.05).

TBARS (mg Eq. MDA/kg W.W.) Phospholipids (g Eq. PC/kg W.W.)

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oxidation interact with other molecules, including proteins, and/or degraded to other substances not accessible to analysis [40], which would explain the shift of TBARS values observed at day 9 at 4 °C. Such decrease in amounts of TBARS was also observed after 9 days of storage of sardines on ice [25]. To this end, we can say that time course of lipid peroxidation in mackerel follows the general pattern of the kinetics of lipid oxidation, in other words; TBARS content increases or decreases in parallel with the decreasing or increasing of hydroperoxides content, respectively. This pattern of evolution is also observed during freezing, where lipid oxidation persists. Moreover, hydroperoxides and TBARS contents are not dependent on mode of storage (P ≥ 0.05). In agreement with Rodriguez et al. [41], it seems that the refrigerated temperature storage is not sufficient to inhibit or slow formation of oxidation products in small pelagic fish. Probably, the acidic pH of these fish would promote activation of haem proteins and release of iron that are pro-oxidant agents [42]. Then, freezing is a good way to increase shelf life of products, because lipid oxidation rate and pro-oxidant effect of haem pigments are reduced at low temperatures. However, it is necessary to reach temperatures of –40 °C to completely stop oxidation, because at a temperature of –15 °C, formation of peroxides stays possible [43].

3.4 Histamine Production

A few hours after capture, initial histamine content in mackerel is already high (6.39 mg/100g). It exceeds the toxic level of histamine of 10 mg/100g muscle rapidly after 24 h of storage at ambient temperature. In agreement with Mendes [44], Ababouch et al. [13] and Chaouqy & El Marrakchi [14], this excessive production is probably due to a high amount of histidine in red muscle of fish (especially the Scombridae), a large proliferation of mesophilic bacteria capable of histidine decaboxylation and an optimum temperature (26 °C) and a suitable pH for

the synthesis and activity of histidine decarboxylase (from 2.5 to 6.5), reached rapidly in the flesh of mackerel [16]. At 4 °C, accumulation of histamine exceeds the threshold, only after 5 days of storage and never at –18 °C (Fig. 5). Indeed, effectiveness of icing in control of histamine production has already been proven on sardine [13], mackerel [16, 44], bonito [45] and other species. Several studies agree that histamine-producing bacteria are mesophilic and at temperatures between 0 and –20 °C, microorganisms are in a slowed down state of life and can not multiply and invade fish muscle [46]. However, other authors [47] have shown that histamine is synthesized at significant levels at temperatures as low as 2-5 °C. Indeed, Photobacterium histaminum and

Photobacterium phosphorum show a decarboxylase

activity at 4 °C and even at –20 °C [48]. However, in frozen mackerel, we see a decrease in histamine content after 2 months of freezing. According to Sato et al. [49], histamine content depends on histamine-producing bacteria as well as on degrading bacteria (bacteria with histaminase activity) [50].

4. Conclusions

Finally, freezing storage has a preserving effect on lipid damage and histamine production and seems the best means of storage; if these species are not consumed during the two days following capture.

The extent of primary lipid oxidation products such

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as hydroperoxides is considered to be one that would provide the most relevant information on the intensity of lipoperoxidation, as long as precautions are taken to prevent their rapid decomposition [51]. In addition, quantifying secondary lipid oxidation products (TBARS), more stable compounds, is considered more reliably to estimate the level of lipid oxidation. By cons, given the bell-shaped curve, it does not determine promptly the level of lipid oxidation [17]. Nevertheless, it seems that the peaks recorded in TBARS correspond to a decrease in hydroperoxides content and very advanced sensory impairments. Therefore, the strategy used, measuring kinetic of phospholipids hydrolysis and lipid oxidation products, appear pertinent for determining; degree of fish lipids oxidation.

Accumulation of histamine is significantly slowed when icing occurs but does not correlate with sensory impairment. By cons, under conditions of storage at ambient temperature, the correlation is good. Indeed, spoilage flora mainly formed by Shewanella

putrefaciens and Pseudomonas that are mesophilic and

psychrophilic, but little or no histidinolytic. At low temperatures, the activity is still possible, so there are many sensory impaired but histamine accumulates in flesh at low levels as long as the Enterobacteriaceae (mesophilic), leading producers of this amine are inhibited [52]. According to Sato et al. [53], Mendes [44] and Chaouqy & El Marrakchi [14], the lack of correlation between accumulation of histamine and sensory impairment, said that monitoring histamine production is more useful as sanitary index rather than spoilage parameter.

For each analysis, the number of specimens collected (n = 5) was not sufficient to avoid changes in biochemical composition between individuals manifested by high standard deviations. It would be preferable and very appropriate to increase number of sample analyzed and make the first analyze on fishing boats to monitor the formation of these compounds from T0 (from capture). This could lead us to a more

accurate estimate of the keeping time of the species studied and to develop a representative index of the real fish freshness.

Acknowledgments

This work was supported by National Agency for a development of research in Health (A.N.D.R.S.). The authors express their gratitude to all members of Laboratory of Aquaculture and Bioremediation (Department of Biotechnology, University of Es-Sénia, Oran, Algeria).

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[32] H. Laghmari, A. El Marrakchi, Appréciation organoleptique et physico-chimique de la crevette rose Parapenaeus longirostris (Lucas, 1846) conservée sous glace et à température ambiante, Revue de Médecine Vétérinaire 156 (2005) 221-226.

[33] J.B. Rossell, Measurement of rancidity, in: J.C. Allen, R.J. Hamilton (Eds.), Rancidity in Foods, Elsevier, New York, 1989, pp. 23-52.

[34] H. Chan, Autooxidation of Unsaturated Lipids, Academic Press, New York, 1987.

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mackerel minces lipid hydrolysis, Journal of Food Science 58 (1993) 79-83.

[36] S.P. Aubourg, Damage detection in horse mackerel (Trachurus trachurus) during chilled storage, Journal of the American Oil Chemists Society 78 (2001a) 857-862.

[37] A.J. De Koning, T.H. Mol, Rates of free fatty acid formation from phospholipids and neutral lipids in frozen cape hake (Merlussius spp) mince at various temperatures, Journal of the Science of Food and Agriculture 50 (1990) 391-398.

[38] G. Özyurt, A. Polat, B. Tokur, Chemical and sensory changes in frozen (–18 °C) wild sea bass (Dicentrarchus labrax) captured at different fishing seasons, International Journal of Food Science and Technology 42 (2007) 887-893.

[39] S.P. Aubourg, Review: Recent advances in assessment of marine lipid oxidation by using fluorescence, Journal of the American Oil Chemists Society 76 (1999) 409-419. [40] S.P. Aubourg, I. Medina, Influence of storage time and

temperature on lipid deterioration during cod (Gadus morhua) and haddock (Melanogrammus aeglefinus) frozen storage, Journal of the Science of Food and Agriculture 79 (1999) 1943-1948.

[41] A. Rodríguez, N. Carriles, J.M. Cruz, S.P. Aubourg, Changes in the flesh of cooked farmed salmon (Oncorhynchus kisutch) with previous storage in slurry ice, LWT-Food Science and Technology 41 (2008) 1726-1732.

[42] C. Genot, A. Meynier, A. Riaublanc, J.M. Chobert, Protein alterations due to lipid oxidation in multiphase systems, in: A. Kamal-Eldin (Ed.), Lipid Oxidation Pathways, AOACS Press Champaign, 2003, pp. 265-292.

[43] P.J. Ke, R.G. Ackman, B.A. Linke, D.M. Nash, Differential lipid oxidation in various parts of frozen mackerel, Journal of Food Technology 12 (1977) 37-47. [44] R. Mendes, Changes in biogenic amines of major

Portuguese bluefish species during storage at different

temperatures, Journal of Food Biochemistry 23 (1999) 33-43.

[45] M.A. Mazorra-Manzano, R. Pacheco-Aguilar, E.I. Diaz-Rojas, M.E. Lugo-Sanchez, Post mortem changes in black skipjack muscle during storage in ice, Journal of Food Science 65 (2000) 774-779.

[46] M.P. Doyle, L.R. Beuchat, T.J. MontVille, Food Microbiology: Fondamentals and Frontiers, ASM Press, Washington DC, 1997, p. 94.

[47] C. Grau, D. Sanchez, A. Zerpa, O. Vallenilla, O. Berti, Study of microflora associated with histamine formation in sardines (Sardinella aurita), Revista Cientifica-Facultad de Ciencias Veterinarias 13 (2003) 199-204.

[48] M. Okuzumi, S. Okuda, M. Awano, Isolation of psychrophilic and halophilic histamine-forming bacteria from Scomber japonicas, Bulletin of the Japanese Society of Scientific Fisheries 47 (1981) 1591-1598.

[49] T. Sato, T. Fujii, T. Masuda, M. Okuzumi, Changes in numbers of histamine-metabolic bacteria and histamine content during storage of common mackerel, Fisheries Science 60 (1994) 299-302.

[50] S.L. Taylor, Histamine food poisoning: Toxicology and clinical aspects, Critical Reviews in Toxicology 17 (1986) 91-128.

[51] M. Laguerre, L.J. Lopez-Giraldo, J. Lecomte, M. Pina, P. Villeneuve, Outils d’évaluation in vitro de la capacité antioxydante, Oléagineux, Corps gras, Lipides 14 (2007) 278-292.

[52] L.H. Ababouch, S.C. Rafaramandimy, V.C. Rahanjavelo, A. Razafindramonjy, A.R. Rabesalama, R. Ducaud, Construction et expérimentation de conteneurs et couffins isothermes pour la pêche artisanale à Madagascar, Les actes de l’Institut Agronomique et Vétérinaire Hassan II 12 (1) (1992) 29-35.

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In Vitro

Regeneration of Commercial Sugarcane

(

Saccharum

spp.) Cultivars in Nigeria

Sani Lawan Abdu1, Mustapha Yahaya2 and Usman Inuwa Shehu3

1. College of Engeneering Science Technology, Federal Polytechnic Kazaure, PMB 5004, Kazuare, Nigeria

2. Department of Biological Sciences, Bayero University Kano, PMB 3011, Kano, Nigeria

3. Department of Plant Science, Institute for Agricultural Research, Ahmadu Bello University, PMB 1044, Zaria, Nigeria

Received: September 11, 2011 / Accepted: November 04, 2011 / Published: July 30, 2012.

Abstract: The effect of different concentrations of 6-benzylaminopurine (BA) with or without 0.2 mg/L NAA on in vitro regeneration of sugarcane (Saccharum spp.) cultivars SP726180, B47419, M1176/77 and M2119/88 were evaluated. Leaf base explants were cultured on Murashige and Skoog (MS) basal medium supplemented with 3.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) for 4 weeks. Thereafter, induction of somatic embryogenesis was observed following the transfer of resulting calli to 2,4-D-free medium for another 4 weeks. Regeneration was achieved by transfer of the embryogenic calli to regeneration media fortified with different concentrations of BA ± α-naphthylacetic acid (NAA). The number, length and vigor of shoots produced in all the genotypes were highest on media supplemented with 1.0 and 1.5 mg/L BA with and without 0.2 mg/L NAA. Among the genotypes tested, B47419 and M1176/77 recorded highest number of shoots, while maximum shoot length and crop vigor was obtained with M1176/77. Induction of callus with 3.0 mg/L 2,4-D and its subsequent incubation on 2,4-D-free media, followed by regeneration on media supplemented with 1.0 or 1.5 mg/L BA with 0.2 mg/L NAA was found to be efficient for in vitro regeneration of the sugarcane genotypes used in this study. This protocol could be applied for micropropagation of other elite genotypes.

Key words: Saccharum spp., leaf explant, 2,4-D, callus, BA, NAA, regeneration.

1. Introduction



Sugarcane (Saccharum spp.) is an important crop for many tropical and subtropical countries which account for aproximately 75% of global sugar production. Efficient photosynthesis and biomass production make this graminae a good candidate for green energy generation and other industrial processing. It is an important cash crop in Nigeria, cultivated for both vegetable and industrial uses. Propagation of sugarcane using stem cutting has always been associated with the problem of transmission of pathogens from one generation to another which results in rapid degradation of improved varieties. Moreover, conventional breeding

Corresponding author: Sani Lawan Abdu, M.Sc., research fields: plant tissue culture and genetic transformation.E-mail: [email protected].

has failed to keep pace with the growing demand for improved varieties, due to limitations imposed by cytogenetic complexity of sugarcane [1].

In vitro propagation offers a faster means of

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embryos are known to be good candidates for genetic transformation of sugarcane, regeneration via somatic embryogenesis is an attractive option for both rapid clonal propagation and genetic transformation of sugarcane. Sugarcane regeneration from callus culture in has been widely reported [3-6]. Since genotype and the type and concentration of exogenous hormones used, tremendously affect morphogenesis in sugarcane and other species, development of genotype specific protocol is critical for rapid propagation of selected sugarcane cultivars. To date no report on successful regeneration of sugarcane cultivars used in Nigeria. This paper report the effect of BA and NAA on in

vitro regeneration of four commercial cultivars in

Nigeria.

2. Materials and Methods

2.1 Plant Materials

Four sugarcane genotypes were used in this study, two (SP726180 and B47/419) were obtained from experimental site of National Sugar Development Company (NSDC), Hadejia. While M2119/88 and M1176/77 were obtained from sugarcane programme of Jigawa Research Institute, Jigawa State, Nigeria. Cane setts were established on screening plots at Jigawa Research Institute, after basal application of Nitrogen fertilizer (100 kg/ha). The plots were watered every other two days and growing tips of six month old plants were used as source of explants. The

in viro study was carried out in the Plant

Biotechnology Laboratory, Jigawa Research Institute, Jigawa State, Nigeria

2.2 Medium and Culture Condition

The meduim used in this study was Murashige and Skoog (MS) [7] basal meduim and was suplemented with 30% sucrose, pH was adjusted to 5.8 with 1 M KOH and solidified with 8% agar before autoclaving for 15 minutes at 121 °C and 15 psi. All cultures were incubated at 29 ± 2 °C.

2.3 Callus Induction and Plant Regeneration

Leaf cylinders provided by immature leaf rolls and apical meristem were used for callus induction. Apical portions of healthy shoots were stripped to the terminal bud and washed with sterilized distilled water and surface sterilized by initial dipping in 70% ethanol (v/v) for 2 minutes followed by treatment with 20% commercial bleach (containing 3.5% sodium hypochlorite w/v) for 20 minutes, thereafter the materials were rinsed 3 times in sterile double distilled water. The outer immature leaf rolls were removed under aseptic condition using a sterile forcep and surgical blade. Explants were cut into 10 mm segments and planted on sterilized calllus induction medium consisting of MS basal medium supplemented with 3.0 mg/L 2,4-D. Ten explants were cultivated in Petri dish (11 mm diameter) containing 10 mL of the callus induction medium and Petri dishes were used per treatment. Cultures were kept in the dark for 4 weeks after which were tranferred to fresh medium for further callus proliferation. After eight weeks of culture in the presence of 2,4-D, the resulting calli were transferred to auxin-free media for another four weeks to induce embryogesis. After 4 weeks of culture on auxin-free media, ramdomized embryogenic callus peices (25 ± 1 mg) were transferred to culture vessels (125 × 25 mm) containing 35 mL of regeneration media consisting of MS meduim modified by addition of 1.0, 1.5 or 2.0 mg/L of BA with or without 0.2 mg/L NAA. Ten vessels were used for each treatment and the experiment was laid in a completely randomized design with 3 replications. Cultures were incubated in growth chamber under 16-hrs photoperiod for eight weeks. The number, length and vigor of the plantlets regenerated for each treatment were recorded.

2.4 Rooting and Acclimatization

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In Vitro Regeneration of Commercial Sugarcane (Saccharum spp.) Cultivars in Nigeria 723

medium supplemented with 60% sucrose and 1.5 mg/L of Indole-butyric acid (IBA). Plantlets with well developed roots were transferred to poly-pots (250 × 125 mm) containing sterilized river sand and manure (2:1) under high humidity (> 90%) created by covering with transparent polythene sheet. The plantlets were watered every day with half-strength MS macro and micro salts. The humidity was reduced gradually by punching hole on the polythene sheet, until it was finally removed after 2 weeks. Acclimatization operations were carried out in the greenhouse.

2.5 Statistical Analysis

The data collected were subjected to analysis of variance (ANOVA) and means were saperated using Duncan Multiple Range Test [8].

3. Result and Discussion

Swelling of explants was observed after 2-3 days of culture (Fig. 1Aa). Callus initiation started after 2 weeks of culture from the cut edges and injured sites and gradually covered the explants (Fig. 1Ab). A close observation of the callus showed two major types; the compact nodular embryogenic callus which appeard white to cream in colour (Fig. 1Ac), and the pale yellow nonembryogenic callus (Fig. 1Ab). Such types of calli were also reported in sugarcane by Ch. Gandonuo et al. [9]. The embryogenic callus eventually differenciated into somatic embryos following 4 weeks of culture on auxin free medium and the embryos developed into shoots by given rise to coleoptile followed by the elongation of the first leaf out of the coleoptile (Fig. 1B). In sugarcane and other monocots, high concentration of 2,4-D is

Fig. 1 A-D: Stages for in vitro regeneration of sugarcane (Saccharum spp.) via somatic embryogenesis. A: callus induction on inmature leaf in the presence of 3.0 mg/L 2,4-D (EP: explant; NEC: non-embryogenic callus; EC: embryogenic callus); B: plantlets regeneration from embryogenic callus (GSE: germinating somatic embryo); C: in viro plantlets; D: acclimatized seedlings in greenhouse.

A

D B

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In Vitro Regeneration of Commercial Sugarcane (Saccharum spp.) Cultivars in Nigeria

724

Table 1 Effect of different concentrations of BA ± NAA on in vitro plantlet regeneration in sugarcane.

PGR Conc. (mg/L)

Shoot number Shoot length Crop vigor Mean ± SD

0.00 8.94 ± 2.54d 2.69 ± 0.63c 2.74 ± 0.87c 0.2 NAA 7.81 ± 0.34e 2.51 ± 0.14cd 1.97 ± 0.15d 1.0 BA 15.10 ± 4.63b 2.83 ± 0.53c 2.78 ± 0.71c 1.5 BA 15.69 ± 3.08ab 2.50± 0.51cd 2.78 ± 0.70c 2.0 BA 12.03 ± 2.51c 2.23 ± 0.42d 1.97 ± 0.74d 1.0BA + 0.2 NAA 16.19 ± 2.56a 4.80 ± 1.34a 4.09 ± 0.96a 1.5BA + 0.2 NAA 15.85 ± 2.26a 4.95 ± 1.01a 3.62 ± 0.87b 2.0BA + 0.2 NAA 12.18 ± 2.31c 3.76 ± 1.22b 3.06 ± 1.13c

CV (%) 14.24 18.05 16.39

required for callus induction and development of bilateral symmetry during enbryogenesis [10, 11]. However, reduction in the concentration of 2, 4-D or its complete removal from the meduim is required for the retention of bilateral symmetry and subsquent expression of somatic embryogenesis [12].

Regeneration was achieved on both hormone-free medium and media fortified with plant growth regulators indicating that once bilateral symmetry is established at callus stage, exogenous hormones are not essential for plantlets regeneration. However, low regeneration frequency was observed on hormone-free medium (Table 1). Addition of exogenous BA ± NAA resulted in an increased frequency of regeneration. Plantlets regeneration was significantly (P < 0.05) higher when MS was supplemented with 1.0-2.0 mg/L BA with or without 0.2 mg/L NAA (Table 1). Regeneration frequency increased with the increase in the concentration of BA from 1.0 to 1.5 mg/L but, further increased in the concentration of BA to 2.0 mg/L caused a decline in the regeneration frequency (Table 1). Plantlets regeneration was significantly (P < 0.05) higher in both number, length and vigor when MS was supplented with 1.5 mg/L BA with 0.2 mg/L NAA, but, addition of 0.2 mg/L NAA alone decreases the frequency of regeneration suggesting a possible synergistic effect of these hormones on morphogenesis in sugarcane. Synergistic effect of combination BA and NAA on plantlets regeneration from callus culture were earlier repoted in sugarcane [13] and wheat [14].

The ratio of cytokinins and auxins proved to be

Fig. 2 Response of sugarcane genotypes to in vitro shoot regeneration.

Fig. 3 Response of sugarcane genotypes to in vitro shoot length and crop vigor, with error bars .

more important with respect to morphogenesis in callus culture of sugarcane. For callus induction and embryogenesis high concentration of auxin is required while high concentration of cytokinins favoured shoots proliferation from callus culture. Although the mechanism of cytikinins is not clearly understood, their ability to initiate shoot from callus in tissue culture suggested thier role in controlling shoot apical meristem development. Kerstetter et al. [15] suggested that regulation of gene expression is one possible mechanism by which cytokinins influence shoot apical meristem development. Increase in cytokinin levels in trangenic Arabidopsis resulted in the increase in the expression STM-genes which regulate shoot apical meristem development [16].

The genotypes used in this study showed variation in their response to in vitro morphogenesis (Figs. 2 and 3). M1176/77 and B47419 exhibited the highest morphogenic potentials, producing significantly (P < 0.05) higher number of well developed shoots than the

Shoot num

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In Vitro Regeneration of Commercial Sugarcane (Saccharum spp.) Cultivars in Nigeria 725

other genotypes tested. Merkle et al. [17] reported that, varation in response to tissue culture among the plant species could be attributed to their physiological differences. The variable responses of these genotypes to the exogenous BA and NAA may be explained by their differences in the levels of endogenous hormones. So, when these genotypes are exposed to exogenous plant growth regulators, their entire hormone levels may be either sub-optimal, optimal or super-optimal for morphogenesis. The response of the genotypes to exogenous plant growth regulators would, therefore, depend mainly on the level of endogenous of hormones in the genotypes which will finally determine their response to in vitro regeneration.

Well developed plantlets (Fig. 1C) were transfered to rooting media consisting of MS supplemented with 1.0 mg/L IBA. Plantlets with well developed roots were transferred to poly-pots (250 × 125 mm) containing sterilized river sand and manure (2:1) under high humidity (> 90%) created by covering the plantlets with transparent polythene sheet. Plantlets were watered every day with half-strength MS (macro and micro salts). Humidity was reduced gradually by punching hole on the polythene sheet, until the sheet was finally removed (Fig. 1D).

In conclusion, Induction of callus with 3.0 mg/L 2,4-D and its subsequent incubation on 2,4-D-free media, followed by regeneration on media supplemented with 1.0 or 1.5 mg/L BA with 0.2 mg/L NAA was found to be efficient for in vitro regeneration of the sugarcane genotypes used in this study. We therefore suggest that this protocol could used to test the response of other sugarcane cultivars to in vitro regeneration.

References

[1] J.A. da Silva, J.A. Bressiani, Sucrose synthase molecular marker associated with sugar content in elite sugarcane progeny, Genetics and Molecular Biology 28 (2005) 294-298.

[2] I.A. Khan, A. Khatri, S. Raza, N. Seema, Nizamani, M.H. Naqvi, Study of genetic variability in regenerated sugarcane plantlets derived from different auxin concentrations, Pak. Sugar. J. 19 (2000) 35-38.

[3] W.J. Ho, I.K. Vasil, Somatic embryogenesis in sugarcane

(Sacharrum officinarum L.), the morphology and ontogeny of somatic embryos, Protoplasm 118 (1983) 169-180. [4] E.A. Brisibe, H. Miyake, T. Taniguchi, E. Maeda,

Regulation of somatic embryogenesis in long term callus cultures of sugarcane (Saccharum officinarum L.), New Phytology 126 (1994) 301-307.

[5] A. Khatri, I.A. Khan, M.A. Javed, M.A. Siddiqui, M.K.R. Khan, M.H. Khanzada, et al., Studies on callusing and regeneration potential of indigenous and exotic sugarcane clones, Asian Journal of Plant Science 1 (2002) 41-43. [6] A.A. Mir Ali, N. Shagufta, F.A. Saddiqui, J. Iqbal, Rapid

clonal multiplication of sugarcane (Saccharum officinarum) through callogenesis and organogenesis, Parkistan Journal of Botany 40 (2008) 123-138.

[7] T. Murashige, F. Skoog, A revised medium for rapid growth and bioassays with tobacco tissue cultures, Physiology Plantae 15 (1962) 473-497.

[8] Statistical Analysis System, User’s Guide Statistics, Version 6 Edition, SAS Institute, Inc. Carry N. C., 1998. [9] Ch. Gandonuo, T. Errabil, J. Abrini, M. Idaomar, F. Chibi,

N.S. Senhaji, Effect of genotype on callus induction and plant regeneration from leaf explants of sugarcane (Saccharum spp.), African Journal of Biotechnology 4 (2004) 1250-1255.

[10] L. Michalczuk, D.M. Ribnicky, T.J. Cooke, J.D. Cohen, Regulation of indole-3-acetic acid biosynthetic pathways in carrot cell cultures, Plant Physiology 100 (1992) 1346-1353. [11] C. Fischer, G. Neuhaus, Influence of auxin on the

establishment of bilateral symmetry in monocots, Plant Journal 9 (1996) 659-669.

[12] V.M. Jimenez, Regulation of in vitro somatic embryogenesis with emphasis on the role of endogenous hormones, Revista Brasiliera de Fisiologia, Vegetal. Vol. 13 (2001) 196-223.

[13] K. Chengalrayan, M. Gallo-meagher, Effect of various growth regulators on shoot regeneration of sugarcane, In Vitro Cellular and Developmental Biology 37 (2001) 434-439.

[14] S. Shrivastava, N. Singh, H.S. Chawla, Role of growth regulators and glutamin for enhancing regeneration response in Wheat (Triticum aestivum L.), Journal of Genetics and Breeding 54 (2000) 71-75.

[15] R.A. Kerstetter, S. Hake, Shoot meristem formation in vegetative development, Plant Cell 9 (1997) 1001-1010. [16] H.M. Rupp, M. Frank, T. Werner, M. Strnad, T.

Schmülling, Increased steady state mRNA levels of the STM and KNATI homeobox genes in cytokinin overproducing Arabidopsis thaliana indicate a role for cytokinins in the shoot apical meristem, Plant Journal 18 (1999) 557-563.

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Validation of Analytical Method of Irbesartan Plasma

in Vitro

by High Performance Liquid

Chromatography-Fluorescence

Harmita, Yahdiana Harahap and I. Kadek Arya M.

Department of Pharmacy, University of Indonesia, Depok, Jakarta 16424, Indonesia

Received: November 29, 2011 / Accepted: February 01, 2012 / Published: July 30, 2012.

Abstract: Irbesartan is an antihypertensive drug whose concentration in blood is very small so it requires a sensitive method of analysis, selective and valid for analysis. In this study, it is carried out optimization of analytical conditions and validation for the analysis of irbesartan in plasma. Chromatography was performed on a C18 column (250 × 4.6 mm, 5 μm) under isocratic elution with acetonitrile-0.1% formic acid (46:54 v/v), pH 3.75. Detection was made at excitation 250 nm and emission 370 nm and analyses were run at a flow-rate of 1.0 mL/min at a temperature of 40 ºC. Losartan potassium was used as internal standard. Plasma extraction was done by deproteination with acetonitrile, mixed with vortex for 30 seconds, then centrifuged it at 10,000 rpm for 10 min. In plasma validation, the recovery was 96.22%, and the lower limit of quantification (LLOQ) in plasma was 2 ng/mL. The method also fulfill the criteria for accuracy and precision intra and inter day by normal values (%Diff) not exceed ± 15%. On the stability study, irbesartan in plasma temperature –20 °C has been stable for 28 days.

Key words: HPLC, fluorescence, irbesartan, validation, human plasma.

1. Introduction



Hypertension is a common health problem in developed countries and developing countries. The cause of hypertension is diverse due to genetic factors, lifestyle, and stress. Uncontrolled hypertension can lead to various diseases such as stroke, heart failure, diabetic nephropathy, myocardial infarction, kidney failure and even death. According to the guidline of the National Joint Committee, the management of patients with hypertension should be precise and fast, with drugs given singly or in combination therapy drugs [1]. Irbesartan is a class of hypertension drugs called angiotensin II receptor blockers that work on the renin-angiotensin-aldosterone system. Besides useful for lowering blood pressure, drugs known as angiotensin II receptor blockers have a protective effect

Corresponding author: Harmita, Ph.D., research field: pharmaceutical chemistry. E-mail: [email protected].

on the kidneys especially in diabetic patients [2]. Irbesartan is a non-peptide compounds, with the chemical name 2-butyl-3-[[29-(1H-tetrazole-5-yl) [1,19-biphenyl]-4yl]methyl]-1,3-diazaspiro[4,4]non-1-en-4-one. Irbesartan is a hypotensive agent that does not require biotransformation to become active form [3]. Peroral drug absorption is rapid, bioavaibilitasnya about 60-80% and 90% protein bound. At therapeutic doses of irbesartan (75-300 mg), maximum concentration in plasma will be obtained about 1.5-2 hours after dosing [4]. The maximum concentration in plasma after administration of a dose of 150 mg irbesartan is about 1.5 ± 0.29 g/mL [5].

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Validation of Analytical Method of Irbesartan Plasma in Vitro by High Performance Liquid Chromatography-Fluorescence

727

chromatography tandem mass spectrometry method (HPLC⁄_MS⁄_{MS) [6-8].}

In bioequivalence studies, the proposed method should be simple and able to process hundreds of samples in a limited time. This paper describes a simple, rapid, precise, and accurate HPLC method for determining irbesartan in human plasma in vitro.

2. Materials and Methods

2.1 Chemicals and Reagents

Irbesartan (99.7% on assay) were obtained from Hetero Labs Limited. Losartan Potassium (99.6% on assay) were obtained Ipca Labs Limited. Acetonitrile and methanol were HPLC-grade and were purchased from Merck. The other chemicals and reagents were analytical grade. Human plasma was provided by Indonesian blood bank (Palang Merah Indonesia).

2.2 Chromatographic Conditions

The HPLC system (Shimadzu, Japan) used consisted of a model LC-10AD pump, a fixed manual injection loop of 20 μL, and a model RF-10AXL Fluorescence detector; data acquisition was performed with the SCL-10A processor. The analytical column employed was a Kromasil C18 column (250 × 4.6 mm, i.d., 5 μm).

The mobile phase consisted of acetonitrile-0.1% formic acid (46:54 v/v). The mobile phase was adjusted to pH 3.75 ± 0.01 with 1 N NaOH or dilute phosphate acid (85% v/v), filtered through a 0.45 μm cellulose membrane filter (Whatman) and degassed before use (Elmasonic S60H). The detection wavelength was set at excitation 250 nm and emission 370 nm. Chromatography separation was performed at temperature 40 °C and flow rate was maintained at 1 mL/min.

2.3 Standard Solutions and Quality Control Samples

Primary stock solutions of irbesartan (1 mg/mL) and losartan potassium (1 mg/mL) were prepared in methanol. Then, diluted with methanol to obtain a

certain concentration.

Human plasma calibration standards of irbesartan were prepared by spiking an appropriate amount of the working standard solutions into drug-free human plasma. The concentration of irbesartan in calibration curve was 2.05 ng/mL, 5.12 ng/mL, 10.24 ng/mL, 51.20 ng/mL, 256 ng/mL, 512 ng/mL, 1,024 ng/mL, and 5,120 ng/mL. Quality control (QC) samples were prepared at three concentrations that were low (6.14 ng/mL), medium (2,048 ng/mL), and high (4,096 ng/mL).

2.4 Sample Preparation

0.25 mL of plasma containing certain concentrations of irbesartan were added 25.0 μL of the internal standard working solution (6 μg/mL). Three part of acetonitril (750.0 μL) was added to precipitate protein in plasma, vortex-mixed for 30 sec and centrifuged at 10,000 rpm for 10 min. A 20 μL aliquot of the supernatant was injected into the HPLC system.

2.5 Validation of This Method

The validation parameters obtained were specificity, linearity, sensitivity, accuracy, precision, recovery and stability. The method was validated according to USFDA guidance for bioanalytical method validation [9].

Six randomly selected blank plasma samples [9] were processed by a similar extraction procedure and analyzed to determine the extent to which endogenous plasma components may contribute to interference at the retention time of irbesartan, and losartan potassium.

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728

were low (6.14 ng/mL), medium (2,048 ng/mL), and high (4,096 ng/mL) concentrations, accompanying by a standard calibration curve on each analytical run.

The recovery of irbesartan was evaluated by comparing measured concentration obtained from peak areas of pre-treated quality control plasma samples (n = 5) with mean measured concentration of those spiked-after extraction samples at the same nominal concentrations. Stability quality control plasma samples were conducted at low and high concentrations and were subjected to short-term (6 h and 24 h) incubation at room temperature, three freeze/thaw cycles, and storage for 28 days (–20 ºC). The stability of primary stock solutions were also being conducted for 25 days (5 ºC).

3. Results

3.1 Specificity

The current method showed excellent chromatographic specificity with no endogenous plasma interference at the retention times of irbesartan and losartan potassium as internal standard. Chromatograms obtained from human blank plasma and human blank plasma spiked with irbesartan (0.5 μg/mL) and losartan potassium (20 μg/mL) are shown in Figs. 1A and 1B, respectively. Irbesartan and

losartan potassium were well resolved with respective retention times of 6.2 min and 10.4 min.

3.2 Calibration Curve and Limit of Quantification

The calibration curves were linear over the concentration range of 2.05-5,120 ng/mL with a correlation coefficient of 0.9999. The correlation coefficient from replicate calibration curves on different days was more than 0.9995. The lower limit of quantification with a coefficient of variation of less than 20% was 2 ng/mL.

3.3 Precision and Accuracy

The coefficient variation values of both inter- and intraday analysis for 5 days at three concentrations which each concentration is conducted at 5 replicates were less than 6.37% whereas the %Diff were less than 13.43%. The inter- and intra-day precision and accuracy values of the assay method are presented in Table 1.

3.4 Recovery

The mean extraction recoveries of irbesartan at three concentrations (low, mid, high) were 86.23%-113.45%, 85.37%-99.31%, and 85.73%- 103.44%, respectively.

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3.5 Stability

Treated plasma samples were found to be stable at least 24 h when the samples were kept at room temperature (%Diff < 15%). The concentrations of irbesartan in plasma which underwent three freeze-thaw cycles or storage at –20 ºC for 28 days were found to be stable with % differentiation less than 15%. The stability data of irbesartan stored under various conditions and subjected to freeze-thaw cycles are shown in Table 2. The primary stock solutions were also found to be stable for 25 days when were kept at 5 ºC. The stability data of irbesartan stored under various conditions and subjected to freeze-thaw cycles are shown in Table 2.

4. Discussion

4.1 Preparation of Plasma Samples

Protein precipitation has the advantages of simplicity and universality, so it was used to prepare the plasma samples [8]. The reason of choosing acetonitrile as the precipitation agent was caused by its ability to precipitate protein, especially when given

not less than the volume of blood. It is also usually become the component of mobile phase with the result that the system will be able to accept. Results indicated that direct protein precipitation with acetonitrile was simple and rapid and good separation of the drug and I.S. was achieved using the precipitation method. The centrifugator was used optimally at 10,000 rpm for 10 min. The aim was to obtain the pure supernatant which was ready to be injected.

4.2 Optimization of Mobile Phase

The chromatographic conditions were optimized by injecting analytes with mobile phase containing varying percentages of organic phase and flow rates of mobile phase to achieve good resolution and symmetric peak shapes for irbesartan and losartan potassium, as well as a short retention time. As expected, the retention times increased with decreasing acetonitrile percentage and system flow rates. The chosen mobile phase pH was 3.75 ± 0.01 by giving the most symmetric peak shapes for irbesartan and losartan potassium.

Table 1 Accuracy and precision from the determination of irbesartan in human plasma (n = 25/concentration).

Concentration Mean ± SD (ng/mL) CV (%) %Diff

Low 5.7972 ± 0.5870 6.85 Min. = –13.77% _{Max. = 14.67%}

Mid 1957.4585 ± 66.6095 3.41 Min. = –14.63% _{Max. = 2.81%}

High 3981.4188 ± 0.0358 4.33 Min. = –14.28%

Max. = 14.51%

Table 2 Stability data of irbesartan in human plasma.

Concentration CV (%) %Diff

Short term stability for 24 h in plasma at room temperature

Low 7.82 –5.79 - 14.46%

High 2.82 –9.54 - 8.11%

Long term storage at –20 °C for 28 days

Low 2.95 –5.94 - 12.84%

High 2.20 –13.94 - 10.47%

Three freeze/thaw cycles

Low 4.64 –14.46 - 9.02%

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730

Fig. 2 Chromatogram of irbesartan (1) and losartan potassium (2) in varying percentages of organic phase. The chromatograms representative system in acetonitrile-0.1% formic acid (46:54, v/v) (A); (37:63, v/v) (B); and (40:60, v/v) (C).

Optimal conditions were a mobile phase consisting of acetonitrile: 0.1% formic acid (46:54, v/v) pH 3.75 ± 0.01 arranged by 1 N NaOH and 85% phosphate acid. Under optimum conditions, the chromatographic run time for each sample was completed within 14 min.

4.3 Advantages of the Method

In comparison to previously published HPLC methods for separation and quantitation of irbesartan, the major modifications incorporated into the current method include: simple sample preparation procedures, common and cheap HPLC equipment and mobile phase additives, and a relative short analysis time as well.

Thus the assay is suitable for routine analysis when determining assay on biological samples to perform bioequivalence studies. A simple, rapid, precise, and accurate HPLC method for determining irbesartan in human plasma has been presented. Although lower sensitivity was obtained in comparison to previously published LC methods with mass spectrometry detection, the resulting LLOQ (2 ng/mL) was sufficient for human pharmacokinetic studies.

5. Conclusion

An analytical method developed for irbesartan quantification in plasma samples showed good specificity, sensitivity, linearity, precision, and accuracy over the entire range of clinically significant

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and therapeutically achievable plasma concentrations, thereby enabling its use in bioequivalence trials.

References

[1] Gilman and Goodman, The Pharmacological Basis of Therapeutics, Chapter 32, 11th ed., The McGraw-Hill Companies, Inc., USA, 2006.

[2] Prakash, Majalah Farmasi, Ed. Juni, Jakarta, 2009.

[3] Martindale, The Complete Drug Reference, Pharmaceutical Press, USA, 2007.

[4] C.F. Lacy, Drug Information Handbook, 13th ed., Lexi Comp., USA, 2000, pp. 826-827.

[5] H.R. Brunner, Hypertens, Lippincott Williams & Wilkins Inc., USA, 1997.

[6] S.K. Bae, J.K. Min, J.S. Eon, Y.C. Doo, HPLC Determination of Irbesartan in Human Plasma: Its Application to Pharmacokinetic Studies, Wiley Interscience, USA, 2008, pp. 568-572, available online at: http://onlinelibrary.wiley.com/doi/10.1002/bmc.1154/abstr act 2.

[7] N. Erk, R. Bischoff, Simultaneous determination of

irbesartan and hydrochlorothiazide in human plasma by liquid chromatography, Departement of Analytical Chemistry, Faculty of Pharmacy, University of Ankara, Turkey, 2002, available online at: http://www. sciencedirect.com/science?_ob=ArticleURL&_udi=B6 X0P-473VRB81&_user=10&_coverDate=01%2F25%2 F2003&_rdoc=1&_fmt=high&_orig=search&_origin=s earch&_sort=d&_docanchor=&view=c&_searchStrId= 1548849389&_rerunOrigin=google&_acct=C00005022 1&_version=1&_urlVersion=0&_userid=10&md5=39f a6759c63b9431803608ed5a1149b4&searchtype=a#m4. com.

[8] N. Ganesan, M. Gomathi, Method development and validation of irbesartan using LCMS/MS: Application to pharmacokinetic studies, A.J. College of Pharmacy, Chennai, India, 2010, available online at: http://jocpr.com/vol2-iss4-2010/ JCPR-2- 4-740-746.pdf. [9] Food and Drug Administration, Guidance for industry: