Development and validation of a reagent based on enzymatic ... · A. Tobeña Montanuy, S. Dueñas...
1
. The presence of histamine in fish is becoming increasingly important to consumers and producers alike, due to the potential threats of toxicity to humans and consequently health implications. In the scientific field, histamine has the potential to be applied as indicator unsanitary conditions during fish manipulation. Current methods for analysis of histamine are HPLC, ELISA, and fluorimetry which sometimes require expensive and sophisticated instrumentation and as consequence skilled technicians. BioSystems presents a new, simple and rapid enzymatic method determination of histamine in fish that can be automated. The method is based on the specific reaction of histamine with recombinant histamine dehydrogenase (from E. Coli) causing the reduction of a soluble tetrazolium salt to form formazan salt that absorbs at 420 nm. Thus can be measured by visible spectrophotometry and correlate it, through a calibration, with histamine concentration. This new reagent has a long stability, is liquid and ready-to-use, avoiding end-user influence. It has been formulated to be used in any photometer or automated analyzer. Linearity, limit of detection (LoD), limit of quantification (LoQ) , repeatability, within laboratory reproducibility, trueness and recovery were characterized using a BioSystems Y15 ® automated analyzer and HPLC method. Method comparison between enzymatic kit & HPLC-UV Type of method: Quantitative Analyte: Histamine Units: mg/kg Matrix: Raw tuna, water-packed tuna, oil-packed tuna, raw sardines, oil-packed sardines, semi-preserved anchovy fillets Range: 10 to 200 mg/kg Reference method used: Analyte, range and units: Histamine, range of 10 to 500 mg/kg Accreditation: PEE/1/119, accredited by ENAC according to UNE-EN ISO/IEC 17025 (Technical Annex No. 96/LE230) Matrix: Fish, aquaculture and derivative products Method: HPLC-UV detector Limits of detection / Limits of quantification DEVELOPMENT AND VALIDATION OF AN ENZYMATIC REAGENT FOR AUTOMATING HISTAMINE ANALYSIS IN FISH Sample preparation (fish): Accurately weigh approximately 5 g of homogenized sample and add 25 ml of distilled water. Shake until the sample is homogeneously suspended. Incubate the sample for 20 minutes in a boiling water bath, stirring periodically. Let stand to room temperature and centrifuge for 10 minutes, at least at 2000g. Histamine in the sample originates, by means of the coupled reactions described below, a colored complex that can be measured by spectrophotometry: 1. M.L. Latorre-Moratalla et al. Validation of an ultra high pressure liquid chromatographic method for the determination of biologically active amines in food. J. Chromatogr. A. 1216 (2009) 7715–7720. 2. Tsuneo Sato et al. Simple and rapid determination of histamine in food using a new histamine dehydrogenase from Rhizobium sp. Anal. Bio- chem. 346 (2005) 320–326. 3. International organization of vine and wine (OIV), Compendium of international methods of wine and must analysis Vol. 1 & 2, Paris (2010). 4. AOAC International (1998). AOAC Peer-Verified Methods Program. Manual on Policies and Procedures. Maryland. USA. INTRODUCTION 5.COMMISSION DECISION (2002/657/EC) of 12 August 2002 implementing Council Directive 96/23/EC concerning the performance of analyti- cal methods and the interpretation of results. 6.COMMISSION DIRECTIVE 98/53/EC of 16 July 1998 laying down the sampling methods and the methods of analysis for the official control of the levels for certain contaminants in foodstuffs. 7.Ellison SLR, Rosslein M, Williams A. (2000). Quantifying uncertainty in analytical measurement. 2nd edition. EURACHEM/CITAC Guide. 8.EURACHEM-GUIDE (1998). The fitness for purpose of analytical methods: A laboratory guide to method validation and related topics. LGC, Teddington. 9.Horwitz W. (1998). A simple method for evaluating data from an interlaboratory study. Journal of AOAC International Vol. 81, No. 6. 10.Hotwitz W. (2003). Validation: An invisible component of measurement. AOAC International. 11.Huber L. (1998). Validation of analytical methods: review and strategy. LC-GC International, pp. 96-105. 12.JC Miller JN. Miller. (1993). Estadística para química analítica. Addison-Wesley Iberoamericana. 2nd edition. 13.Statistical Subcommittee of the AMC. (2004). The amazing Horwitz function. Technical Brief No. 17 July. Royal Society of Chemistry. 14.Thompson M. (2000). Recent trends in inter-laboratory precision at ppb and sub-ppb concentrations in relation to fitness for purpose criteria in proficiency testing. Analyst, 125 385-386. 15.Thompson M, Ellison S. LR, Wood R. (2002). Harmonized guidelines for single-laboratory validation of methods of analysis. IUPAC Technical Report. Pure Appl. Chem., vol 74, no. 5, pp. 835,855. Abstract Product Performance Measurement procedure Conclusions Bibliography Reagents and calibrators are ready to use and stable until expiry date. Results confirms that FoodQuality ® Histamine Enzymatic (BioSystems) can quantitatively measure histamine in fish samples with similar accuracy and precision than the HPLC method. Furthermore, this test can be automated in a analyzer (the kit is adapted for Y15 ® or Y25 ® analyzers from BioSystems) or can be used in a photometer with a simple sample treatment making histamine analysis more accurate, fast and easy. The small volumes used also allows an affordable analysis and better waste management. A. Tobeña Montanuy, S. Dueñas Ros, S. Muñoz Hernández BioSystems S.A., Barcelona (Spain) [email protected] Raw Mackerel Raw Sardine Pickled anchovies ANFACO (HPLC) 34 39 64 61 BioSystems 10 12 16 14 97 89 Canned Tuna 2 2 8 8 121 122 567 592 450 437 Anchovies paté 4 4 0 0 Tuna sirloin 65 74 7 7 Canned Sardine 28 27 1 0 Canned Mackerel 69 81 50 25 0 -25 -50 0 100 200 300 Histamine (mg/Kg) % Diference 400 500 600 50 25 0 -25 -50 0 100 200 300 Histamine (mg/Kg) % Diference 400 500 600 600 500 400 300 200 100 0 0 100 200 300 mg/Kg (ANFACO) mg/Kg (BioSystems) 400 500 600 Oil-packed sardines LOD = 5.77 2.85 4.28 4.58 0.29 7.01 17 8.5 13 14 0.9 21 LOQ = 3 x LOD Semi-preserved anchovy fillets Water-packed Tuna Oil-packed Tuna Raw Tuna Raw Sardines Best-fit values Slope Y-int. when X=0.0 0.9774 ± 0.01387 2.332 ± 2.647 95% CI Slope Y-int. when X=0.0 0.9476 to 1.007 -3.345 to 8.009 Goodness of fit r 0.9972 Data Number of X values 17 Equation y = 0.9774x + 2.332
Transcript of Development and validation of a reagent based on enzymatic ... · A. Tobeña Montanuy, S. Dueñas...
.
The presence of histamine in fish is becoming increasingly important to consumers and producers alike, due to the potential threats of toxicity to humans and consequently health implications. In the scientific field, histamine has the potential to be applied as indicator unsanitary conditions during fish manipulation. Current methods for analysis of histamine are HPLC, ELISA, and fluorimetry which sometimes require expensive and sophisticated instrumentation and as consequence skilled technicians. BioSystems presents a new, simple and rapid enzymatic method determination of histamine in fish that can be automated.
The method is based on the specific reaction of histamine with recombinant histamine dehydrogenase (from E. Coli) causing the reduction of a soluble tetrazolium salt to form formazan salt that absorbs at 420 nm. Thus can be measured by visible spectrophotometry and correlate it, through a calibration, with histamine concentration. This new reagent has a long stability, is liquid and ready-to-use, avoiding end-user influence. It has been formulated to be used in any photometer or automated analyzer. Linearity, limit of detection (LoD), limit of quantification (LoQ) , repeatability, within laboratory reproducibility, trueness and recovery were characterized using a BioSystems Y15® automated analyzer and HPLC method.
Method comparison between enzymatic kit & HPLC-UV
Type of method: QuantitativeAnalyte: HistamineUnits: mg/kgMatrix: Raw tuna, water-packed tuna, oil-packed tuna, raw sardines, oil-packed sardines, semi-preserved anchovy filletsRange: 10 to 200 mg/kg
Reference method used:Analyte, range and units: Histamine, range of 10 to 500 mg/kgAccreditation: PEE/1/119, accredited by ENAC according to UNE-EN ISO/IEC 17025 (Technical Annex No. 96/LE230)Matrix: Fish, aquaculture and derivative productsMethod: HPLC-UV detector
Limits of detection / Limits of quantification
DEVELOPMENT AND VALIDATION OF AN ENZYMATIC REAGENT FOR AUTOMATING HISTAMINE ANALYSIS IN FISH
Sample preparation (fish): Accurately weigh approximately 5 g of homogenized sample and add 25 ml of distilled water. Shake until the sample is homogeneously suspended.Incubate the sample for 20 minutes in a boiling water bath, stirring periodically. Let stand to room temperature and centrifuge for 10 minutes, at least at 2000g.
Histamine in the sample originates, by means of the coupled reactions described below, a colored complex that can be measured by spectrophotometry:
1. M.L. Latorre-Moratalla et al. Validation of an ultra high pressure liquid chromatographic method for the determination of biologically active amines in food. J. Chromatogr. A. 1216 (2009) 7715–7720.2. Tsuneo Sato et al. Simple and rapid determination of histamine in food using a new histamine dehydrogenase from Rhizobium sp. Anal. Bio-chem. 346 (2005) 320–326.3. International organization of vine and wine (OIV), Compendium of international methods of wine and must analysis Vol. 1 & 2, Paris (2010).4. AOAC International (1998). AOAC Peer-Verified Methods Program. Manual on Policies and Procedures. Maryland. USA. INTRODUCTION5.COMMISSION DECISION (2002/657/EC) of 12 August 2002 implementing Council Directive 96/23/EC concerning the performance of analyti-cal methods and the interpretation of results.6.COMMISSION DIRECTIVE 98/53/EC of 16 July 1998 laying down the sampling methods and the methods of analysis for the official control of the levels for certain contaminants in foodstuffs.7.Ellison SLR, Rosslein M, Williams A. (2000). Quantifying uncertainty in analytical measurement. 2nd edition. EURACHEM/CITAC Guide.8.EURACHEM-GUIDE (1998). The fitness for purpose of analytical methods: A laboratory guide to method validation and related topics. LGC, Teddington.9.Horwitz W. (1998). A simple method for evaluating data from an interlaboratory study. Journal of AOAC International Vol. 81, No. 6.10.Hotwitz W. (2003). Validation: An invisible component of measurement. AOAC International.11.Huber L. (1998). Validation of analytical methods: review and strategy. LC-GC International, pp. 96-105.12.JC Miller JN. Miller. (1993). Estadística para química analítica. Addison-Wesley Iberoamericana. 2nd edition.13.Statistical Subcommittee of the AMC. (2004). The amazing Horwitz function. Technical Brief No. 17 July. Royal Society of Chemistry.14.Thompson M. (2000). Recent trends in inter-laboratory precision at ppb and sub-ppb concentrations in relation to fitness for purpose criteria in proficiency testing. Analyst, 125 385-386.15.Thompson M, Ellison S. LR, Wood R. (2002). Harmonized guidelines for single-laboratory validation of methods of analysis. IUPAC Technical Report. Pure Appl. Chem., vol 74, no. 5, pp. 835,855.
Abstract Product Performance
Measurement procedure
Conclusions
Bibliography
Reagents and calibrators are ready to use and stable until expiry date.
Results confirms that FoodQuality® Histamine Enzymatic (BioSystems) can quantitatively measure histamine in fish samples with similar accuracy and precision than the HPLC method. Furthermore, this test can be automated in a analyzer (the kit is adapted for Y15® or Y25® analyzers from BioSystems) or can be used in a photometer with a simple sample treatment making histamine analysis more accurate, fast and easy. The small volumes used also allows an affordable analysis and better waste management.
A. Tobeña Montanuy, S. Dueñas Ros, S. Muñoz HernándezBioSystems S.A., Barcelona (Spain)
Raw Mackerel
Raw Sardine
Pickled anchovies
ANFACO (HPLC)
34 39
6461
BioSystems
10 12
1614
9789
Canned Tuna
2 2
88
121122
567592
450437
Anchovies paté 44
00Tuna sirloin
6574
77Canned Sardine
2827
10Canned Mackerel
6981
50
25
0
-25
-500 100 200 300
Histamine (mg/Kg)
% D
ifere
nce
400 500 600
50
25
0
-25
-500 100 200 300
Histamine (mg/Kg)
% D
ifere
nce
400 500 600
600
500
400
300
200
100
00
100 200 300mg/Kg (ANFACO)
mg/
Kg
(Bio
Sys
tem
s)
400 500 600
Oil-packed sardines
LOD = 5.77 2.85 4.28 4.58 0.29 7.01
17 8.5 13 14 0.9 21LOQ = 3 x LOD
Semi-preservedanchovy fillets
Water-packedTuna
Oil-packedTuna
RawTuna
RawSardines
Best-fit valuesSlopeY-int. when X=0.0
0.9774 ± 0.013872.332 ± 2.647
95% CISlopeY-int. when X=0.0
0.9476 to 1.007-3.345 to 8.009
Goodness of fitr 0.9972
DataNumber of X values 17
Equation y = 0.9774x + 2.332
