DEVELOPMENT AND VALIDATION OF A REAGENT BASED ON
ENZYMATIC REACTIONS FOR DETERMINING HISTAMINE IN WINES
A. Tobeña Montanuy, S. Dueñas Ros, S. Muñoz Hernández , N. Toro Funes
BioSystems S.A., Barcelona (Spain)
[email protected]
Abstract
Product performance
The presence of histamine in wine 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 of
unsanitary conditions during wine production procedures.
Precision
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 for
determination of histamine in red and white wine.
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 a
formazan salt that absorbs at 420 nm. Thus can be measured by visible spectrophotometry
and correlate it, through a calibration, with histamine concentration. A simple method to
remove interfering substances (reducing agents like polyphenols and anthocyanins) from
wine sample is also presented.
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, withinlaboratory reproducibility, trueness and recovery were characterized using a BioSystems
Y15® automated analyzer and HPLC method OIV-MA-AS315-18 as the reference.
Evaluation of precision performance of quantitative measurement methods; Approved guideline Second edition. CLSI EP5.
Samples: control materials with low and high histamine concentration.
Results are expressed as coefficient of variation (%CV).
Repeatability
Between-run
Within-laboratory
13 mg/L
0,8 %
1,7 %
1,9 %
Limit of detection (LoD)/quantification (LoQ)
Establish the LoD and/or LoQ of a measurement procedure; Approved guideline - Second edition.
CLSI EP17-A2.
Limit of Detection (LoD)
1 mg/L
Limit of Quantification (LoQ)
3 mg/L
Measurement range
Measurement range
Measurement procedure
Histamine in the sample originates, by means of the coupled reactions described below, a
colored complex that can be measured by spectrophotometry:
8 mg/L
1,3 %
1,7 %
2,2 %
3 - 80 mg/L
Trueness
Red and white commercial wines from Spain where spiked with histamine to reach different
concentration levels. Samples were tested using BioSystems FoodQuality® Histamine Enzymatic
according to the instructions with Y15® automated analyzer from BioSystems. The same samples
were assayed with UHPLC according to the Latorre et al. protocol, an optimization of OIV-MAAS315-183. All samples where assayed (extraction and test) in duplicate.
Data is analyzed using Passing-Bablok linear regression and Bland-Altman difference analysis
with Analyse-it® software.
Sample preparation: Dilute wine ¼ with distilled water.
Add approximately 10 mg of PVPP and shake.
Centrifuge sample to obtain a firm sediment and use the supernatant.
Samples are assayed in a Y15® which automatically are dispensed with the reagents in the
reaction rotor. Absorbance at 420 nm is read and concentration is calculated through a 5
points calibration.
Reagents and calibrators are ready to use and stable until expiry date.
Conclusions
Best-fit values
Slope
1,018 ± 0,01898
Y-int. when X=0.0
0,8362 ± 0,4303
1/slope
0,9822
95% CI
Slope
0,9801 to 1,056
Y-int. when X=0.0
-0,02667 to 1,699
Goodness of Fit
R square
0,9812
Data
Number of X values
57
Equation
Y = 1,018*X + 0,8362
Results confirms that FoodQuality® Histamine Enzymatic (BioSystems) can quantitatively
measure histamine in wine samples with similar accuracy and precision than the UHPLC
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.
Bibliography
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. Biochem. 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).
www.admeo.us
Recovery
The average recovery for all the wines is 97% (86 – 109%). There are no difference between the
type or origin of wine (red or white).
www.biosystems.es