Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 151 (2015) 861–866
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Short Communication
Rapid and sensitive method for analysis of nitrate in meat samples using ultra performance liquid chromatography–mass spectrometry Masoom Raza Siddiqui a,⇑, Saikh Mohammad Wabaidur a, Zeid A. ALOthman a, M.Z.A. Rafiquee b,c a
Advanced Materials Research Chair, Chemistry Department, College of Science, King Saud University, P.O. Box 2455, 11451 Riyadh, Saudi Arabia Chemistry Department, College of Science, King Saud University, P.O. Box 2455, 11451 Riyadh, Saudi Arabia c Department of Applied Chemistry, Aligarh Muslim University, Aligarh 202002, India b
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
UPLC–MS method has been proposed
UPLC–MS chromatogram of nitrate in analyzed meat sample and its corresponding spectrum (m/z = 62).
for the determination of nitrate. Developed method was applied for nitrate estimation in meat samples. The method utilizes a surfactant and an organic modifier as a mobile phase. Recovery of the nitrate in the meat samples were in the range of 98.02– 98.99%.
a r t i c l e
i n f o
Article history: Received 21 October 2014 Received in revised form 30 June 2015 Accepted 6 July 2015 Available online 7 July 2015 Keywords: Ultra performance liquid chromatography Mass spectrometry Nitrate Meat
a b s t r a c t A sensitive and selective ultra performance liquid chromatography–mass spectrometric method has been developed for the quantitative analysis of nitrate in meat samples. Selected ion reaction (SIR) mode was adopted to identify and quantify the nitrate. Chromatographic analyses were performed on a BEH C-18 column with a mobile phase consisting of a surfactant (Cetylpyridinium chloride) and acetonitrile in equal ratio (50/50, v/v) at a flow rate of 0.4 mL min1. The limit of detection and limit of quantitation of the developed method was found to be 0.0599 and 0.1817 mg kg1, respectively. The linearity of the proposed method was checked in the concentration range of 0.5–10 mg kg1 with an excellent correlation coefficient (r) of 0.997. The recovery of the nitrate in the meat samples were in the range of 98.02–98.99%. Ó 2015 Published by Elsevier B.V.
1. Introduction Nitrate is a polyatomic ion having molecular formula NO 3 . It is a common natural compound and normally present in soils, water and food. In recent times, large amount of nitrates are produced for the agricultural purpose, such as fertilizers, owing to its high ⇑ Corresponding author. E-mail address: [email protected] (M.R. Siddiqui). http://dx.doi.org/10.1016/j.saa.2015.07.028 1386-1425/Ó 2015 Published by Elsevier B.V.
solubility and possible biodegradability. However, besides its agricultural use, another major application is found in the meat industry where it is applied to control the growth of the pathogens thus contributing in its preservation [1]. Several reports have appeared in the literature highlighting the usage of nitrate salts of sodium and potassium (E251 and E 252) are used as preservative to protect against Clostridium botulinum and other species of Clostridium [2]. Sodium salt of nitrates is key additive in the cured meat industry to give the meat a salty and better taste; other reasons for the
862
M.R. Siddiqui et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 151 (2015) 861–866
addition of nitrate to the meat are color fixation and indirect beneficial effect on its flavor. Barbieri et al. reports nitrate exerts an antioxidant action against the fats and is believed to stabilize the red color of the meat [3]. Despite of the several applications in the meat industry, nitrates are reported to be one of the toxic components from food, whose poisoning can lead to discoloration of the blood due to presence of methemoglobin, which is oxygen-carrying metalloprotein hemoglobin [4,5]. Further, nitrates in the oral cavities and in the stomach gets converted into nitrite, which can reacts with the amines and amides from the amino acids to form carcinogenic N-nitroso compounds [6]. The alarm about the nitrate was emerged in about middle of the 20th century when Comly reported the case of blue baby syndrome which led to the origin of the regulation of nitrate content [7]. European parliament and council directive on food additive [8] permits ingoing amount of 300 mg kg1 of nitrate in most of the meat products while the same for the residual amounts were 250 mg kg1. In a similar type of regulation, USA in their Code of Federal Regulations limits the amount of nitrate to not more than 500 mg kg1 in the finished meat products. Food Standard agency, U.K. permits 150 mg kg1 of nitrate in meat product category. Many articles have been reported concerning analytical aspect of nitrate in various matrices [9–12]. In the literatures it is observed that all the regulations, laws and directives consider nitrate as toxic substance when taken in excess amount. Several methods have been developed to quantitate nitrate in meat including HPLC [13–20], spectrophotometry [21–25], potentiometric [26,27], capillary electrophoresis [28–30]. Full automated Robotic method has been reported for the estimation of nitrate in cured meat sample [31]. To the best of our knowledge, there is no ultra performance liquid chromatography–mass spectrometry (UPLC–MS) method available in the literature for the determination of nitrate in meats samples. Thus this experiment was aim at developing a rapid method to determine the quantity of nitrate in meat samples using UPLC–MS because of its rapid analyses and excellent separations ability for food analysis [32,33].
Cetylpyridinium chloride and acetonitrile (50:50, v/v) at a flow rate of 0.4 mL min1. Equal volume (5 lL) of both the standard and the test samples were injected into the chromatograph individually for the quantitative analysis purpose. The nitrate samples were allowed to run for 1.0 min and the peak for the nitrate appeared at 0.65 min. 2.4. Mass spectrometric condition The identification and determination of nitrate was done using a Micromass Quattro Premier triple–quadrupole mass spectrometer fitted with an ESI source. Primary vacuum to the mass spectrometer was provided by Oerlikon rotary pump; model Sogevac SV40 BI (Paris, France). The mass spectrometer was functioned at negative ionization mode to generate ion at m/z 62 for nitrate. The monitoring conditions were also optimized. The parameters optimized were capillary voltage 3.0 kV, cone voltage 40 V, source temperatures 120 °C and desolvation temperature 300 °C. The cone gas flow and the desolvation gas flow were set at 60 L h1 and 600 L h1. Highly pure (99.99%) nitrogen was used during the mass spectrometric analysis, desolvation and cone gas was produced by a Peak Scientific NM30LA nitrogen generator (Inchinann, UK). 2.5. Preparation of standard stock solution and test samples 2.5.1. Standard solution preparation Standard stock solution of nitrate was prepared by dissolving 1000 mg of sodium nitrate in 1000 mL of Milli Q water. Working standard solutions were prepared by further diluting the standard stock solution.
Chemicals and the solvents were of HPLC or analytical grade. Sodium nitrate was obtained from Sigma–Aldrich, Germany. Milli Q purified water was used throughout the experiment. Cetylpyridinium chloride was procured from Sigma–Aldrich. Standards and test samples were filtered through 0.45 lm PVDF syringe filters before injecting into the chromatograph.
2.5.2. Sample preparation The non-spiked and the processed meat samples (10 g) were mixed, homogenized and 100 mL of Milli Q water was added to it. The whole content was placed at 80 °C for 15 min and then it was allowed to cool at room temperature. The content was then centrifuged for 10 min at 6000 rpm at room temperature and the supernatant was filtered through Whatman filter paper then prior to the chromatographic analysis the filtered samples were again filtered through 0.45 lm PVDF syringe filters (Membrane Solutions, Texas USA). The recovery sample were prepared in four sets, each set consists of three nitrate free meat samples. To each sample a predetermined quantity of nitrate was added all the samples were allowed to stand for 15 min so that the nitrate is absorbed by the meat samples. Further, similar extraction and separation procedure was adopted to determine nitrate in the spiked samples.
2.2. Instrumentation and analytical condition
3. Result and discussion
The determination of nitrate was performed using Water’s Acquity ultra-performance liquid chromatography system (Waters Corp., Milford, MA, USA) equipped with a mass detector. The data processing for quantification of standard and the test samples were carried out using MassLynx™ V4.1 software (Waters Corp., Milford, MA, USA). UPLC BEH C18 column (100 mm 2.1 mm, 1.7 lm particle size) were used for the separation of nitrate, the chromatographic analyses were carried out at 25 °C.
The development of a high throughput method for the determination of nitrate was essential as there is no any UPLC–MS method reported for the determination of nitrate in meat samples. The developed method has shown an improvement in terms performance over the existing methods. Short analysis time, low solvent consumption and a good sensitivity were the priorities of the work. To obtain the best result optimization of various experimental conditions were performed and are mentioned below.
2. Material and methods 2.1. Chemicals and reagents
3.1. Optimization of chromatographic condition 2.3. Chromatographic condition Under the optimized condition the chromatographic peak of nitrate was obtained on BEH C18 column (100 mm 2.1 mm, 1.7 lm particle size) using a binary mobile phase mixture of
Nitrate content was estimated using LC–MS on C18 column using a Cetylpyridinium chloride (CPC) and acetonitrile as organic modifier. CPC is a cationic surfactant and has been used in this study because as it do not produce substantial environmental
M.R. Siddiqui et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 151 (2015) 861–866
threat [34]. It shows nontoxic behavior at lower concentration (20 10
NA NA 1.0–2.9
82–117 103–114 >90
14 15 17
HPLC Spectrophotometry Spectrophotometric Spectrophotometry Flow injection spectrophotometric method Capillary electrophoresis
Meat products Meat Meat products Meat and vegetable Food stuffs including meat Meat products and vegetables Food products Meats cured meat Meat samples
75–94 ppm 9 mg kg1 9 lg L1
0–150 mg kg1 4.5 500 lg kg1– 3.75 g kg1 0–250 lg of solute 0.5–20 mg L1 0.034–3.95 mg L1 10–100 ppm 1–10 mg L1
15 30 >120 30 5
NA 2.1–2.9 NA NA NA
88–93 92–94 NA NA 93–110
18 19 20 21 24
5 106– 1 104 Mol L1 0.5–100 mg L1 69.9–198.1 mg kg1 0.5–30 mg L1 0.5–10 mg kg1
3
2.54
92–106
27
5 5 20 1
0.8–3.7 0.8–1.1 1.31 1.69–1.97
90–109 96.9–101.9 NA 98.02–98.99
28 29 30 This method
Capillary electrophoresis Capillary electrophoresis Flow injection spectrophotometric UPLC–MS
20 mg kg1 0.099 lg mL1 0.3 mg L1 0.16 mg L1 NA 0.0599 mg kg1
NA – data not available.
2 h sample preparation step and 30 min of analysis time [21]. Other procedure involving flow injection analysis has reported 20 mg kg1 LOD and a recovery of 93–110%. [24]. Capillary electrophoretic method reported a LOD of 0.3 mg L1 and a recovery of 90–109% [28]. In an attempt to estimate nitrate in brain tissues Jovanovic et al., achieved 0.4 lmol L1 LOD, while the retention time was 5.3 min [39]. In the present work, UPLC–MS combine technique was used to achieve a faster separation and low analysis time of 1 min. The LOD and LOQ of 0.029 and 0.088 mg kg1 were achieved due to the high sensitivity and selectivity of the MS detector. The results obtained indicate that very low level of the nitrate could be quantitated in the meat samples and meets the sensitivity required by various regulatory authorities. A detailed comparison of the developed method with the existing methods has been mentioned in Table 6. 4. Conclusion The developed UPLC–MS method offers a rapid and accurate and robust quantitative method for nitrate determination processed meat samples. No matrix interference was observed during the experiments and the obtained results show that the method has excellent recoveries. The method is free from extensive cleanup procedure except for the preparation of the meat sample. To the best of our knowledge this is the first UPLC–MS method for the determination of nitrate in meat samples. Moreover, low solvent consumption is an added advantage of the proposed technique. Thus the proposed method is suitable for the determination of nitrate in meat sample and can be used for the routine analysis of the nitrate. Acknowledgements The authors extend their sincere appreciation to the Deanship of Scientific Research at King Saud University for its funding this Prolific Research Group (PRG-1436-04). References [1] R. Michalski, I. Kurzyca, Pol. J. Environ. Stud. 15 (2006) 5–18. [2] I.M.P.L.V.O. Ferreira, S. Silva, Talanta 74 (2008) 1598–1602. [3] G. Barbieri, M. Bergamaschi, G. Barbieri, M. Franceschini, Meat Sci. 93 (2013) 283–286.
[4] M. Iammarino, A.D. Taranto, Int. J. Food Sci. Technol. 47 (2012) 1852–1858. [5] D. Vlascici, O. Bizerea., E. Fagadar-Cosma, C. Brumaru, G. Papoe. Comparaive study for determination of nitrate in meat products, in: ISIRR 2003, section IV, Hunedoara, Romania. [6] J.M. Maanen, A.A. van Geel, J.C. Kleinjans, Cancer Detect. Prev. 20 (1996) 590– 596. [7] H.H. Comly, J. Am. Med. Assoc. 257 (1987) 2788–2792. [8] European Parliament and Council Directive No 95/2/EC of 20 February 1995 on food additives other than colors and sweeteners (OJ L 61, 18.3.1995, p. 1). [9] M.R. Khan, Z.A. Othman, M.A. Khan, R. Busquets, I.H. Alsohaimi, Anal. Met. 5 (2013) 1225–1230. [10] M. Pandurangappa, Y. Venkataramanappa, Anal. Met. 3 (2011) 1838–1844. [11] D. Cogan, J. Cleary, T. Phelan, E. McNamara, M. Bowkett, D. Diamond, Anal. Met. 5 (2013) 4798–4804. [12] B. Hafezi, M.R. Majidi, Anal. Met. 5 (2013) 3552–3556. [13] L. Merino, U. Edberg, G. Fuchs, P. Aman, J. AOAC Int. 83 (2000) 365–375. [14] M. Wootton, S.H. Kok, K.A. Buckle, J. Sci. Food Agric. 36 (1985) 297–304. [15] J.P. De Kleijn, K. Hove, Analyst 109 (1984) 527–528. [16] E. Bianchi, R. Bruschi, R. Draisci, L. Lucentini, Z. Lebensm.-Unters. Forsch. 200 (1995) 256–260. [17] D.C. Siu, A. Henshall, J. Chromatogr. A 804 (1998) 157–160. [18] P.E. Jackson, P.R. Haddad, S.J. Dilli, J. Chromatogr. A 295 (1984) 471–478. [19] A. Alonso, B. Etxaniz, M.D. Martinez, Food Add. Contam. 9 (1992) 111–117. [20] S.M. Oliveira, T.I.M.S. Lopes, A.O.S.S. Rangel, J. Food Sci. 69 (2004) C690–C695. [21] N.A. Zatar, M.A. Abu-Eid, A.F. Eid, Talanta 50 (1999) 819–826. [22] A.A. Ensafi, M. Keyvanfard, Anal. Lett. 27 (1994) 169–182. [23] J.F. Van Staden, T.A. Van der Merwe, S. Afr, J. Chem. 51 (1998) 109–113. [24] R. Andrade, C.O. Viana, S.G. Guadagnin, F.G.R. Reyes, S. Rath, Food Chem. 80 (2003) 597–602. [25] R. Pérez-Olmos, R. Herrero, J.L.F.C. Lima, M.C.B.S.M. Montenegro, Food Chem. 59 (1997) 305–311. [26] K.K. Choi, K.W. Fung, Analyst 105 (1980) 241–245. [27] N. Öztekin, M.S. Nutku, F.B. Erim, Food Chem. 76 (2002) 103–106. [28] C. Merusi, C. Corradini, A. Cavazza, C. Borromei, P. Salvadeo, Food Chem. 120 (2010) 615–620. [29] E.A. Pereira, J.F. Da Silveira Petruci, A.A. Cardoso, Food Anal. Met. 5 (2012) 637– 642. [30] A. Velasco-Arjona, J.A. Garcı´a-Garrido, R. Quiles-Zafra, M.D. Luque de Castro, Talanta 46 (1998) 969–976. [31] M.J. Moorcroft, J. Davis, R.G. Compton, Talanta 54 (2001) 785–803. [32] A.L.S. Dias, E. Rozet, G. Chataigné, A.C. Oliveira, C.A.S. Rabelo, P. Hubert, J. Chromatogr. B 907 (2012) 108–116. [33] M.C. Díaz-García, J.M. Obón, M.R. Castella, J. Collado, M. Alacid, Food Chem. 138 (2013) 938–949. [34] D.A. Edwards, D.T., Wasan, in: D.T. Wasan, M.E. Ginn, D.O. Shah (Eds.), Surfactant in Chemical/Process Engineering, Surfactant Science Series, vol. 28, Marcel Dekker inc, New York, 1988, p. 82. [35] National Library of Medicine Toxicology Data Network. . [36] A. Mohammad, N. Jabeen, Acta Chromatogr. 13 (2003) 135–153. [37] N. Nakov, J. Acevska, K. Brezovska, R. Petkovska, A. Dimitrovska, Maced. J. Chem. Chem. En. 31 (2012) 47–54. [38] C.J. Briscoe, M.R. Stiles, D.S. Hage, J. Pharm. Biomed. Anal. 44 (2007) 484–491. [39] M.C. Jovanovic, M. Djukic, I. Vasiljevic, M.N. Inkovic, M. Jovanovic, J. Serb. Chem. Soc. 72 (2007) 347–356.
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Short Communication
Rapid and sensitive method for analysis of nitrate in meat samples using ultra performance liquid chromatography–mass spectrometry Masoom Raza Siddiqui a,⇑, Saikh Mohammad Wabaidur a, Zeid A. ALOthman a, M.Z.A. Rafiquee b,c a
Advanced Materials Research Chair, Chemistry Department, College of Science, King Saud University, P.O. Box 2455, 11451 Riyadh, Saudi Arabia Chemistry Department, College of Science, King Saud University, P.O. Box 2455, 11451 Riyadh, Saudi Arabia c Department of Applied Chemistry, Aligarh Muslim University, Aligarh 202002, India b
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
UPLC–MS method has been proposed
UPLC–MS chromatogram of nitrate in analyzed meat sample and its corresponding spectrum (m/z = 62).
for the determination of nitrate. Developed method was applied for nitrate estimation in meat samples. The method utilizes a surfactant and an organic modifier as a mobile phase. Recovery of the nitrate in the meat samples were in the range of 98.02– 98.99%.
a r t i c l e
i n f o
Article history: Received 21 October 2014 Received in revised form 30 June 2015 Accepted 6 July 2015 Available online 7 July 2015 Keywords: Ultra performance liquid chromatography Mass spectrometry Nitrate Meat
a b s t r a c t A sensitive and selective ultra performance liquid chromatography–mass spectrometric method has been developed for the quantitative analysis of nitrate in meat samples. Selected ion reaction (SIR) mode was adopted to identify and quantify the nitrate. Chromatographic analyses were performed on a BEH C-18 column with a mobile phase consisting of a surfactant (Cetylpyridinium chloride) and acetonitrile in equal ratio (50/50, v/v) at a flow rate of 0.4 mL min1. The limit of detection and limit of quantitation of the developed method was found to be 0.0599 and 0.1817 mg kg1, respectively. The linearity of the proposed method was checked in the concentration range of 0.5–10 mg kg1 with an excellent correlation coefficient (r) of 0.997. The recovery of the nitrate in the meat samples were in the range of 98.02–98.99%. Ó 2015 Published by Elsevier B.V.
1. Introduction Nitrate is a polyatomic ion having molecular formula NO 3 . It is a common natural compound and normally present in soils, water and food. In recent times, large amount of nitrates are produced for the agricultural purpose, such as fertilizers, owing to its high ⇑ Corresponding author. E-mail address: [email protected] (M.R. Siddiqui). http://dx.doi.org/10.1016/j.saa.2015.07.028 1386-1425/Ó 2015 Published by Elsevier B.V.
solubility and possible biodegradability. However, besides its agricultural use, another major application is found in the meat industry where it is applied to control the growth of the pathogens thus contributing in its preservation [1]. Several reports have appeared in the literature highlighting the usage of nitrate salts of sodium and potassium (E251 and E 252) are used as preservative to protect against Clostridium botulinum and other species of Clostridium [2]. Sodium salt of nitrates is key additive in the cured meat industry to give the meat a salty and better taste; other reasons for the
862
M.R. Siddiqui et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 151 (2015) 861–866
addition of nitrate to the meat are color fixation and indirect beneficial effect on its flavor. Barbieri et al. reports nitrate exerts an antioxidant action against the fats and is believed to stabilize the red color of the meat [3]. Despite of the several applications in the meat industry, nitrates are reported to be one of the toxic components from food, whose poisoning can lead to discoloration of the blood due to presence of methemoglobin, which is oxygen-carrying metalloprotein hemoglobin [4,5]. Further, nitrates in the oral cavities and in the stomach gets converted into nitrite, which can reacts with the amines and amides from the amino acids to form carcinogenic N-nitroso compounds [6]. The alarm about the nitrate was emerged in about middle of the 20th century when Comly reported the case of blue baby syndrome which led to the origin of the regulation of nitrate content [7]. European parliament and council directive on food additive [8] permits ingoing amount of 300 mg kg1 of nitrate in most of the meat products while the same for the residual amounts were 250 mg kg1. In a similar type of regulation, USA in their Code of Federal Regulations limits the amount of nitrate to not more than 500 mg kg1 in the finished meat products. Food Standard agency, U.K. permits 150 mg kg1 of nitrate in meat product category. Many articles have been reported concerning analytical aspect of nitrate in various matrices [9–12]. In the literatures it is observed that all the regulations, laws and directives consider nitrate as toxic substance when taken in excess amount. Several methods have been developed to quantitate nitrate in meat including HPLC [13–20], spectrophotometry [21–25], potentiometric [26,27], capillary electrophoresis [28–30]. Full automated Robotic method has been reported for the estimation of nitrate in cured meat sample [31]. To the best of our knowledge, there is no ultra performance liquid chromatography–mass spectrometry (UPLC–MS) method available in the literature for the determination of nitrate in meats samples. Thus this experiment was aim at developing a rapid method to determine the quantity of nitrate in meat samples using UPLC–MS because of its rapid analyses and excellent separations ability for food analysis [32,33].
Cetylpyridinium chloride and acetonitrile (50:50, v/v) at a flow rate of 0.4 mL min1. Equal volume (5 lL) of both the standard and the test samples were injected into the chromatograph individually for the quantitative analysis purpose. The nitrate samples were allowed to run for 1.0 min and the peak for the nitrate appeared at 0.65 min. 2.4. Mass spectrometric condition The identification and determination of nitrate was done using a Micromass Quattro Premier triple–quadrupole mass spectrometer fitted with an ESI source. Primary vacuum to the mass spectrometer was provided by Oerlikon rotary pump; model Sogevac SV40 BI (Paris, France). The mass spectrometer was functioned at negative ionization mode to generate ion at m/z 62 for nitrate. The monitoring conditions were also optimized. The parameters optimized were capillary voltage 3.0 kV, cone voltage 40 V, source temperatures 120 °C and desolvation temperature 300 °C. The cone gas flow and the desolvation gas flow were set at 60 L h1 and 600 L h1. Highly pure (99.99%) nitrogen was used during the mass spectrometric analysis, desolvation and cone gas was produced by a Peak Scientific NM30LA nitrogen generator (Inchinann, UK). 2.5. Preparation of standard stock solution and test samples 2.5.1. Standard solution preparation Standard stock solution of nitrate was prepared by dissolving 1000 mg of sodium nitrate in 1000 mL of Milli Q water. Working standard solutions were prepared by further diluting the standard stock solution.
Chemicals and the solvents were of HPLC or analytical grade. Sodium nitrate was obtained from Sigma–Aldrich, Germany. Milli Q purified water was used throughout the experiment. Cetylpyridinium chloride was procured from Sigma–Aldrich. Standards and test samples were filtered through 0.45 lm PVDF syringe filters before injecting into the chromatograph.
2.5.2. Sample preparation The non-spiked and the processed meat samples (10 g) were mixed, homogenized and 100 mL of Milli Q water was added to it. The whole content was placed at 80 °C for 15 min and then it was allowed to cool at room temperature. The content was then centrifuged for 10 min at 6000 rpm at room temperature and the supernatant was filtered through Whatman filter paper then prior to the chromatographic analysis the filtered samples were again filtered through 0.45 lm PVDF syringe filters (Membrane Solutions, Texas USA). The recovery sample were prepared in four sets, each set consists of three nitrate free meat samples. To each sample a predetermined quantity of nitrate was added all the samples were allowed to stand for 15 min so that the nitrate is absorbed by the meat samples. Further, similar extraction and separation procedure was adopted to determine nitrate in the spiked samples.
2.2. Instrumentation and analytical condition
3. Result and discussion
The determination of nitrate was performed using Water’s Acquity ultra-performance liquid chromatography system (Waters Corp., Milford, MA, USA) equipped with a mass detector. The data processing for quantification of standard and the test samples were carried out using MassLynx™ V4.1 software (Waters Corp., Milford, MA, USA). UPLC BEH C18 column (100 mm 2.1 mm, 1.7 lm particle size) were used for the separation of nitrate, the chromatographic analyses were carried out at 25 °C.
The development of a high throughput method for the determination of nitrate was essential as there is no any UPLC–MS method reported for the determination of nitrate in meat samples. The developed method has shown an improvement in terms performance over the existing methods. Short analysis time, low solvent consumption and a good sensitivity were the priorities of the work. To obtain the best result optimization of various experimental conditions were performed and are mentioned below.
2. Material and methods 2.1. Chemicals and reagents
3.1. Optimization of chromatographic condition 2.3. Chromatographic condition Under the optimized condition the chromatographic peak of nitrate was obtained on BEH C18 column (100 mm 2.1 mm, 1.7 lm particle size) using a binary mobile phase mixture of
Nitrate content was estimated using LC–MS on C18 column using a Cetylpyridinium chloride (CPC) and acetonitrile as organic modifier. CPC is a cationic surfactant and has been used in this study because as it do not produce substantial environmental
M.R. Siddiqui et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 151 (2015) 861–866
threat [34]. It shows nontoxic behavior at lower concentration (20 10
NA NA 1.0–2.9
82–117 103–114 >90
14 15 17
HPLC Spectrophotometry Spectrophotometric Spectrophotometry Flow injection spectrophotometric method Capillary electrophoresis
Meat products Meat Meat products Meat and vegetable Food stuffs including meat Meat products and vegetables Food products Meats cured meat Meat samples
75–94 ppm 9 mg kg1 9 lg L1
0–150 mg kg1 4.5 500 lg kg1– 3.75 g kg1 0–250 lg of solute 0.5–20 mg L1 0.034–3.95 mg L1 10–100 ppm 1–10 mg L1
15 30 >120 30 5
NA 2.1–2.9 NA NA NA
88–93 92–94 NA NA 93–110
18 19 20 21 24
5 106– 1 104 Mol L1 0.5–100 mg L1 69.9–198.1 mg kg1 0.5–30 mg L1 0.5–10 mg kg1
3
2.54
92–106
27
5 5 20 1
0.8–3.7 0.8–1.1 1.31 1.69–1.97
90–109 96.9–101.9 NA 98.02–98.99
28 29 30 This method
Capillary electrophoresis Capillary electrophoresis Flow injection spectrophotometric UPLC–MS
20 mg kg1 0.099 lg mL1 0.3 mg L1 0.16 mg L1 NA 0.0599 mg kg1
NA – data not available.
2 h sample preparation step and 30 min of analysis time [21]. Other procedure involving flow injection analysis has reported 20 mg kg1 LOD and a recovery of 93–110%. [24]. Capillary electrophoretic method reported a LOD of 0.3 mg L1 and a recovery of 90–109% [28]. In an attempt to estimate nitrate in brain tissues Jovanovic et al., achieved 0.4 lmol L1 LOD, while the retention time was 5.3 min [39]. In the present work, UPLC–MS combine technique was used to achieve a faster separation and low analysis time of 1 min. The LOD and LOQ of 0.029 and 0.088 mg kg1 were achieved due to the high sensitivity and selectivity of the MS detector. The results obtained indicate that very low level of the nitrate could be quantitated in the meat samples and meets the sensitivity required by various regulatory authorities. A detailed comparison of the developed method with the existing methods has been mentioned in Table 6. 4. Conclusion The developed UPLC–MS method offers a rapid and accurate and robust quantitative method for nitrate determination processed meat samples. No matrix interference was observed during the experiments and the obtained results show that the method has excellent recoveries. The method is free from extensive cleanup procedure except for the preparation of the meat sample. To the best of our knowledge this is the first UPLC–MS method for the determination of nitrate in meat samples. Moreover, low solvent consumption is an added advantage of the proposed technique. Thus the proposed method is suitable for the determination of nitrate in meat sample and can be used for the routine analysis of the nitrate. Acknowledgements The authors extend their sincere appreciation to the Deanship of Scientific Research at King Saud University for its funding this Prolific Research Group (PRG-1436-04). References [1] R. Michalski, I. Kurzyca, Pol. J. Environ. Stud. 15 (2006) 5–18. [2] I.M.P.L.V.O. Ferreira, S. Silva, Talanta 74 (2008) 1598–1602. [3] G. Barbieri, M. Bergamaschi, G. Barbieri, M. Franceschini, Meat Sci. 93 (2013) 283–286.
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