Application note: A10-011A The Effect of Filter Selection on the LOD in the Fluorometric Determination of Histamine in Seafood

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Edith Richardson
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1 Fluorimeter 627/ Application note: A1-11A The Effect of Filter Selection on the in the Fluorometric Determination of Histamine in Seafood Introduction Histamine is a biogenic amine that can be formed in food when histidine, a naturally occurring amino acid, is converted to histamine by the metabolic processes of microorganisms. Consumption of foods that contain high levels of biogenic amines can result in an illness called histamine poisoning, where symptoms may range from a mild rash and sweating to severe nausea and vomiting (1). As a result, the maximum permitted level of histamine in seafood is set at 15mg/kg in the European Union and this level is reduced to 5mg/kg in the USA. Many foods can support significant histamine formation; fermented products, such as salami, cheese, and canned sauerkraut have been shown to have histamine concentrations high enough to cause illness. However, the major sources of dietary biogenic amines include several species of fish such as tuna and mackerel, which are known to contain large amounts of free histidine in their muscle tissue (1). Analytical methods such as the fluorometric method described in the AOAC official method Histamine in Seafood, have a low detection threshold called the limit of detection (). The can be determined from the limiting signal-to-noise ratio, where the signal, S, is the difference between readings obtained with the sample and blank solutions, and N is the standard deviation of the blank response. The is given by the instrument readings that give a signal equal to three times the standard deviation of the noise. = (sample conc / S) x (N x 3) Factors other than noise that can affect the concentration corresponding to the include the spectral bandwidth of the excitation and emission filters, the intensity of the exciting light that can be concentrated on the sample, the fraction of the fluorescence collected by the detection system, the response time of the detection system and the purity of the solvent. The size and arrangement of the sample container with respect to the light beams are also important, as they affect both the desired signal and the extraneous signal that only contributes noise. This application note will use different combinations of narrow and wide bandpass excitation and emission filters to determine the concentration of histamine corresponding to the. The application note will also consider the relative sensitivity and suitability of the model 627 fluorimeter with a silicon diode detector and the model which contains a photomultiplier tube (PMT) when performing the AOAC Histamine in Seafood methodology. Materials The following aqueous solutions were prepared:.1m HCl (8.6ml of 36% hydrochloric acid to 1ml); 1.M NaOH (4.g in 1ml); 1.2M H 3PO 4 (12.2 ml of 85% phosphoric acid to 1ml); 1% solution of orthophthalate aldehyde (.1g to 1ml). A stock solution of histamine hydrochloride was prepared by transferring.837g of histamine hydrochloride into a 5ml volumetric flask and filling to volume with.1m HCl. This solution contained 1mg/ml of histamine. A series of dilute standards were prepared by diluting aliquots of the stock solution with.1m HCl to give solutions with concentrations of 1μg/ml, 5μg/ml, 1μg/ml, 5μg/ml, 1μg/ml and.5μg/ml. Disposable plastic cuvettes (part code 6 247) were used for all measurements. Method The level of histamine in fish and their derived products is commonly determined using the methodology described in the AOAC official method (2). This method prescribes the preparation of a series of standards ranging in concentration from.1 to 1μg/ml histamine. These standard solutions correspond to sample concentrations in the range 5 to 5mg/kg. The histamine-opa reaction product has excitation and emission maxima at approximately 35nm and 45nm respectively. Therefore the fluorimeters were fitted with either the UG nm bandpass filter (part code ) or the 35nm narrow bandpass filter (part code ) for excitation and either the 46nm narrow bandpass filter (part code ) or the BG nm bandpass filter (part code ) for emission. Although the maximum emission intensity for the histamine-opa reaction product occurs at approximately 45nm, the results obtained using the available 46nm filter should closely match those that would have been obtained using the 45nm filter due to the broad peak width of the emission spectrum of the histamine-opa reaction product as shown in Figure 1. Tel:

2 Fluorescence Intensity The difference between the average signal of the sample,, and the average signal of the blank solution,, gave the difference, S, which is the net signal due to the histamine. The was then calculated as follows: = (sample concentration / S) x (SD x 3) Wavelength (nm) Figure 1: Fluorescence emission spectrum of the histamine-opa reaction product (3) The instrument has a % gain setting which can be adjusted to alter the sensitivity of the instrument s detector. The % gain was set using an aliquot of 1μg/ml standard placed in the sample cell and then adjusting the % gain so that the fluorescence signal from the standard was approximately 9% of the full range of the instrument..5ml aliquots of each of the diluted standard solutions had 1.ml of.1m HCl,.3ml of 1M NaOH solution and.1ml of orthophthalate aldehyde added. The solutions were mixed and after 4 min,.3ml of phosphoric acid was added. After not more than 3 min the emission was measured. A blank solution, prepared by using an aliquot of.1m HCl in place of the diluted standard solution, was placed in the sample cell and ten independent readings were measured by removing and reinserting the cuvette after each reading. The average of these ten readings,, was used in the final calculation. The blank was then replaced with the highest concentration standard from the linear response range (R 2.995). Ten independent readings of the standard solution were measured by again removing and reinserting the cuvette after each reading. The average of these ten signals,, was used in the final calculation. This procedure was repeated on both Jenway fluorimeters (627 and ) with each combination of excitation and emission filters being tested to enable a comparison of the detection limits. The value of the signal noise for each combination of filters was obtained by calculating the standard deviation of the emission signal as follows: SD = where: = mean of the series of readings = value of the individual reading n = number of readings. Results Excitation filter UG1 (32-38nm), Emission filter BG28 (38 5nm) summary of the results is shown in Table 1 and Figures 2A and 2B. To obtain an approximate 9% full scale fluorescence reading for the 1μg/ml standard solution the % gain on the was set at 34% Blank Table 1: Fluorescence intensity results for the UG1 excitation filter and the BG28 emission filter 2A 2B R 2 =.8598 R 2 = ( - 5ug/ml) ( - 5ug/ml) R 2 =.9994 R 2 = ( - 1ug/ml) ( - 1ug/ml) Figures 2A and 2B: The linearity of the fluorescence response using the UG1 excitation filter and the BG28 emission filter Tel:

3 From the data the 1μg/ml solution was selected as this was the highest concentration standard that gave a linear response (R 2 >.995) on both the 627 and instruments. instrument and the calculated values are shown in Table 2. The had an of 7.75ng/ml and that of the 627 had an approximately 7 times higher at 54.26ng/ml. 627 Blank 1μg/ml Blank 1μg/ml Mean SD Table 2: Histamine results and determination for the UG1 excitation filter and the BG28 emission filter Excitation filter UG nm, Emission filter Narrow bandpass 46nm summary of the results is shown in Table 3 and Figures 3A, 3B and 3C. To obtain an approximate 9% full scale fluorescence reading for the 1μg/ml standard solution the % gain on the was set at 44% Blank Table 3: Fluorescence intensity results for the UG1 excitation filter and the 46nm emission filter 3A R 2 =.9375 R 2 = ( - 5ug/ml) ( - 5ug/ml) 3B 3C R 2 =.9948 R 2 = ( - 1ug/ml) ( - 1ug/ml) R 2 = ( - 5μg/ml) Figures 3A, 3B and 3C: The linearity of the fluorescence response using the UG1 excitation filter and the 46nm emission filter From the data the 1μg/ml solution was selected for the 627 and the 5μg/ml solution was selected for the instrument as these were the highest concentration standards that gave a linear response (R 2 >.995) on each instrument. instrument and the calculated values are shown in Table 4. The had an of.34ng/ml and the 627 had an approximately 85 times higher at ng/ml. 627 Blank 1μg/ml Blank 5μg/ml Mean SD Table 4: Histamine results and determination for the UG1 excitation filter and the 46nm emission filter Excitation filter Narrow bandpass 35nm, Emission filter Narrow bandpass 46nm summary of the results is shown in Table 5 and Figures 4A, 4B and 4C. Tel:

4 To obtain an approximate 9% full scale fluorescence reading for the 1μg/ml standard solution the % gain on the was set at 69% Blank Table 5: Fluorescence intensity results for the 35nm excitation filter and the 46nm emission filter 4A 4B 4C R 2 =.926 R 2 = ( - 5ug/ml) ( - 5ug/ml) R 2 =.9886 R 2 = ( - 1ug/ml) ( - 1ug/ml) R 2 = ( - 5μg/ml) Figures 4A, 4B and 4C: The linearity of the fluorescence response using the 35nm excitation filter and the 46nm emission filter From the data collected the 1μg/ml standard was selected for the 627 instrument and the 5μg/ml Tel: standard was selected for the instrument as these were the highest concentration standards that gave a linear response (R 2 >.995) on each instrument. instrument and the calculated values are shown in Table 6. The had an of.43ng/ml and the 627 had an approximately 8 times higher at ng/ml. 627 Blank 1ug/ml Blank 5ug/ml Mean SD Table 6: Histamine results and determination for the 35nm excitation filter and the 46nm emission filter Table 7 shows a summary of the values for each combination of filters on each instrument. The wide bandpass filter combination gave the lowest value on the 627 whilst using a wide bandpass filter for the excitation wavelengths and a narrow bandpass filter for the emission wavelengths gave the lowest value on the. However the for the 627 is far higher than the on the indicating that the instrument with the PMT detector has a higher sensitivity. 627 Excitation Emission filter filter UG1 BG UG1 46nm nm 46nm Table 7: Summary of the calculated values for filter combinations under test Conclusions Of the three filter combinations tested on the 627 the wide bandpass filter combination gave the lowest. This is due to the respective bandwidths of these filters allowing more energy to be absorbed by the sample and a greater proportion of the fluorescence signal emitted by the sample to be detected by the 627. In contrast, this filter combination gave the highest on the as the increased sensitivity of the photomultiplier tube (PMT) detector meant that the detected signal was subject to a significant amount of background noise. When a narrow bandpass filter was used as the emission filter, the value on the 627 increased significantly and the fluorescence signal from the histamine-orp product became virtually undetectable when the narrow bandpass excitation filter was also used. In contrast, when a wide or narrow bandpass excitation filter was used in combination with the narrow bandpass emission filter, the values on the were virtually identical. This is due to the adjustable gain setting on this model which allows the

5 sensitivity of the detector to be adjusted to compensate for the reduction in fluorescence signal. The calculated values become significant when compared to the maximum levels of histamine that are permitted in foods such as fresh fish and other derived products. A maximum level of 15mg/kg is specified is the European Union and this level is reduced to 5mg/kg in the USA (4). As a dilution factor of 5 is applied to the measured histamine values in the AOAC method (2) this gives the working values shown in Table 8 for the 627 and fluorimeters, when used for the determination of histamine in seafood. (4) Alasalvar C and Taylor T. Seafoods - Quality, Technology and Nutraceutical Applications. (22) Springer: Excitation filter Emission filter Histamine in Seafood Working (mg/kg) UG1 BG UG1 46nm nm 46nm Table 8: Summary of the working values in the AOAC method for the filter combinations under test Table 8 shows that the 627 values for the UG1/46nm and 35nm/46nm filter combinations are greater than the 5mg/kg maximum permitted level in the United States. The UG1/BG28 filter combination gave an value below this level but as Jenway recommends that customers should not use wide bandpass filters in applications where samples could contain interfering compounds, this combination is also considered to be unsuitable for the determination of histamine in seafood according to the AOAC official method. The data in Table 8 for the shows that all three filter combinations give values below 5mg/kg. Based on the data in this study, Jenway recommends that a fluorimeter, fitted with narrow bandpass filters for excitation and emission, is used when determining the level of histamine according to the AOAC official method Histamine in Seafood. References (1) Farid E. Ahmed. Seafood Safety. (1991) Institute of Medicine (U.S.). Committee on Evaluation of the Safety of Fishery Products: 95. (2) AOAC official method Histamine in Seafood, Fluorometric Method. (3) Kazuo K, Eizou H, Kiyohisa U, Junko K and Yoshihiro T. (1994). Quantification of Histamine by Postcolumn Fluorescence Detection High-Performance Liquid Chromatography Using Orthophthalaldehyde in Tetrahydrofuran and Reaction Mechanism. Analytical Sciences: Vol.1, No.2, Tel:

Spectrophotometer. An instrument used to make absorbance, transmittance or emission measurements is known as a spectrophotometer :

Spectrophotometer. An instrument used to make absorbance, transmittance or emission measurements is known as a spectrophotometer : Spectrophotometer An instrument used to make absorbance, transmittance or emission measurements is known as a spectrophotometer : Spectrophotometer components Excitation sources Deuterium Lamp Tungsten