Total Cyanide measurement in cassava

and other food products

Abstract:
This experiment is focus on determining the level of cyanide in food products using picrate
method with a Bradbury K2 kit and a modified method. The experimental finding of cyanide
levels in almonds is 3-14 part per million (ppm) or 3-14 milligram hydrogen cyanide per
kilogram of almond. 600-900ppm cyanide concentration for apple seeds, 900-1400ppm for
apricot kernel and 6-400ppm for cassava chips, none of those food products can will lethal
damage to humans if consumes in small amounts.
The experimental result shows a large degree of difference between different method of
interoperate of data, ranging from Bradbury colour chart, spectrometer absorbance reading
multiply by 396 and standard calibration curve calculation from absorbance reading. Future
improvement of the experiment procedure is required to better perform the experiment and
achieve a better result.
Introduction:
Cyanide ion is a toxic and lethal substance to humans when ingested and inhaled, it
commonly present in our daily nutrient intakes such as almonds, apple seeds, apricot kernel
and cassava chips. The four-food product all contain either Amygdalin or Linamarin[1].
Linamarin is rapidly hydrolysed to glucose and acetone cyanohydrin, under the natural or
alkaline condition, then it is decomposed to acetone and HCN/CN-. Under the alkaline
condition of the gut linamarin, acetone cyanohydrin and HCN/CN- is all decomposition into
CN-. Cyanide is a rapidly acting, toxic chemical that can be absorbed by inhalation and
ingestion, with extensive amount of cyanide in body, in could lead to the toxic effect by
mechanism including inhibiting cytochrome c oxidases, resulting in cellular hypoxia and
cytotoxic anoxia and eventually result in death[2]. The Lethal Dose to 50% population LD50
for gaseous hydrogen cyanide is 100-300 ppm will result death within 10 to 60-minute,
inhalation of 2000ppm will cause death within 1 minute. The LD50 for ingestion is 50-200mg
or 1 to 3 mg/kg of body weight[3].
The level of cyanide presence in the food product can be reduced to a safe level for human
consumption, Food processing procedures such as soaking, drying and fermentation reduce
cyanide level by the use of plant enzyme and leaching. Granting, soaking, fermentation and
storage will conversion of cyanogenic glycosides to cyanide, with exposure to air or water
cyanide is dissipate out of the food, therefor make it safe for consummation[4].
In this experiment, the aims are to determine the level of cyanide in the four-food product
using Picrate method with Bradbury B2 kit and a modified picrate method similar to the
Bradbury B2 kit. Comparing the result of both methods and to the literature values of others
journal article.

Method:
Part A:
Prepare 100mg sample of cassava chips, apricot kernels, almonds and apple seeds, placed the
powder into Bradbury B2 kit and let it stand for 16-24 hours with caps closed. Prepared a
standard 50mg and a blank sample for validation. Read off from the colour chart for total
cyanide content in ppm in food product. Dissolve Picrate paper in test tube with 5ml
deionised water and leave for 30 minutes at room temperature. Zero the spectrometer with
black standard and measures the absorbance of the other picrate solution. Total cyanide
content is 396 X Absorbance.
Part B:
Prepare using a 30mm * 10mm strip of picrate paper attached to a 25mm * 70mm plastic
strip with PVA glue. Prepare a series of Amygdalin standard solution from 0, 5, 10, 50, 100,
200, 500 ppm and a series of Linamarin standard solution from 0, 200, 500ppm using 0.1M
phosphoric acid in a 30mL plastic bottle. Measure 100mg of four food product and add
500ml of 0.1M phosphoric acid in a 30ml bottle and perform in triplicate. Add 20 mL4M
NaOH to all bottles with 2mL of 0.1M phosphate buffer. Add 50ml enzyme solution to all
bottles and leave it stand for 16-24 hours. Dissolve Picrate paper in test tube with 5ml
deionised water and leave for 30 minutes at room temperature. Zero the spectrometer with
black standard and measures the absorbance of the other picrate solution. Total cyanide
content is 396 X Absorbance. Plot a standard calibration curve and calculate the total cyanide
content in ppm.
Exact procedure can be find in the laboratory by Fu Shalin for Forensic Toxicology. [5]

Result and Discussion

The result for almond in both part A and part B shows a large difference between different
way of interoperating the data. In part A, using the Bradbury kit it has a concentration of 5
part per million (ppm) and a result of 11.880 ppm using spectrometer, it has a difference of
137.6%. The difference might be cause by misreading the colour on the colour chat, as in
lower concentration the colour chart has similar colour though out the range with slight
variance. In part B, it has an average concentration of 3.33ppm using the Bradbury chart,
9.990ppm using the spectrometer and 13.13ppm using the standard calibration method. It still
shows a large degree of difference between difference method to interoperate the data. The
literature value for almond has a average of 25.20 ppm for sweet almonds, the difference
between the experimental value and the literature value may due to the species of almond use.
[6]
The result for apple seed in both part of the experiment shows a similar result in
concentration reading. IN part A, it has a concentration of 800ppm and 744.48ppm using the
two method of data analysis with a difference of 6.94%. In part B, it shows the result in a
range of value between 600pppm to 900ppm, the subjective result of 800ppm using Bradbury
chart is in the centre of the others, but still provide a good estimate of cyanide level. The
literature value for an apple seed is 610 ppm [7], the experimental values is close to the
literature values.

Apricot kernel contains the highest amount of cyanide in all 4-food sample tested. The
Bradbury chart cannot provide an accurate estimate of the cyanide level, instead it only
provides an information that apricot kernel has a greater then 800ppm concentration of
cyanide in its system. With the analysis of sample in both part A and B using the
spectrometer, is has a result of 990ppm and 920.360ppm respectively, which is a close value.
There is a large difference when comparing the result to a standard calibration method of
45%. The literature value of 851ppm suggest the spectrometer method has a closer value to
the literature value.
Cassava Chip contains linamarin, in part A and B is has a similar value using the
spectrometer of 21.780ppm and 20.988ppm respectively, but a significant difference in the
Bradbury chart of 6.67ppm and 30ppm, which the 6.67ppm concentration is a outlier in the
result, this might due to inaccurate comparison using the Bradbury colour chart, the standard
calibration curve shows a largest value of 36.14ppm. The literature value of cyanide is 13-
27ppm of cyanide in cassava [8], it is close to the experimental value around 20-37ppm.
Table 1: Part A result

Samples: Concentration using Concentration using % Difference

Bradbury Chat (ppm) spectrometer (ppm)
Blank 0 0 0%
Standard 50 34.452 31.10%
Almond 5 11.880 -137.60%
Apple Seed 800 744.48 6.94%
Apricot 800+ 990.00 Cannot Determined
Cassava Chip 30 21.780 27.40%

Table 2: Part B result

Samples: Concentration using Concentration using Concentration using

Bradbury Chart (ppm) spectrometer (ppm) standard calibration
curve (ppm)
Almond 3.33 9.900 13.1369
Apple Seed 800 609.840 914.9226
Apricot 800+ 921.360 1383.177
Cassava Chip 6.67 20.988 36.14286

The standard calibration method in part B, has an equation of line of y=0.0168x+0.0293 for
amygdalin and a coefficient of determination of 0.9842, it demonstrate the calibration is a
linear function, the equation of line for linamarin is y=0.0014x+0.0024 and a coefficient of
determination of 0.9903, it also shows a linear function in this calibration. The calibration for
linamarin should be improve in future experiment by adding more data to the calibration
curve, as currently is only offers 2 point and a blank for the construction of calibration. Both
calibration curve should be discarded as both standard calibrations does not meet it own
concentration of hydrogen cyanide level added, providing a large difference in reading when
compare to other method of data interoperation.
 Absorbance verse Standard HCN equivalents
0.9
0.8 f(x) = 0.02 x + 0.03
R² = 0.98
0.7
0.6
Absorbance

0.5
0.4
0.3
0.2
0.1
0
0 10 20 30 40 50 60
Standard HCN equivalents (mg)

Graph 1: Absorbance verse mass of Standard HCN for Amygdalin

Absorbance verse Standard HCN equivalents

0.08
f(x) = 0.03 x − 0.04
0.07 R² = 1
0.06
0.05
Absorbance

0.04
0.03
0.02
0.01
0
0.5 1 1.5 2 2.5 3 3.5 4 4.5
mass of HCN equivalents (mg)

Graph 2: Absorbance verse mass of Standard HCN for Linamarin

Conclusion:
The experiments result finds the level of cyanide in almonds, apple seeds, apricot kernel and
cassava chips are 3-14ppm, 600-920ppm, 900-1400ppm and 6-40ppm respectfully. Through
out the result a large difference can be seen across the whole range of food products tested. In
future practical a increase of sample size is required to get a result in a smaller range and the
standard calibration for this experiment was completed poorly with a result all higher then the
expected values.
Answer to Question:
1. Both enzymes use in this experiment allows the breakdown cyanogenic compounds,
in part A Linamarase are responsible for the breakdown of linamarin into hydrogen
cyanide. In part b, Amygdalase are responsible for the breakdown of amygdalin into
hydrogen cyanide. Both enzymes are chemical selective catalyst, it will only break
down cyanogenic compound into hydrogen cyanide and will break any other chemical
structures.
 2. Part A could have introduced repetition for the samples to be tested, in part B standard
calibration for linamarin would required few more data point to improve the linearity
of the curve and increase it validity.
3. The Bradbury Kit method would not be adequate to be used as a quality assurance
protocols for the manufacturing and consumption of cassava chips, it is only a
subjective view on the results and many errors can be produce during the reading off
the chart. The use of Spectrometer combined with a standard calibration would be
adequate for the propose of quality assurance protocol, it allows an exact value to be
determined under the use of a standard calibration curve. In order to use the method
for quality assurance, the calibration must have a coefficient of determination close to
1 and not less then 0.99.
Reference:
[1] New Zealand Food Safety Authority, Cyanogenic Glycosides- Information Sheet, viewed
24/03/2019,
<https://www.foodsafety.govt.nz/elibrary/industry/Cyanogenic_Glycosides-
Toxin_Which.pdf>.
[2] Bhandari, R.K. et al. 2014, 'Cyanide toxicokinetics: the behavior of cyanide, thiocyanate
and 2-amino-2-thiazoline-4-carboxylic acid in multiple animal models', Journal of
analytical toxicology, vol. 38, no. 4, pp. 218-25.
[3] International Cyanide Management Code 2018, Environmental & Health Effect viewed 24
March 2019, <https://www.cyanidecode.org/cyanide-facts/environmental-health-
effects>.
[4] Food Standards Australia & New Zealand 2019, Cassave and bamboo shoots, viewed
19th March 2019,
<http://www.foodstandards.gov.au/consumer/chemicals/cassava/Pages/default.aspx>.
[5] Forensic Toxicology 2019, 'Practical Manual ', University of Technology Sydney.
[6] Chaouali, N. et al. 2013, 'Potential Toxic Levels of Cyanide in Almonds (Prunus
amygdalus), Apricot Kernels (Prunus armeniaca), and Almond Syrup', ISRN
toxicology, vol. 2013, pp. 610648-.
[7] Holzbecher, M.D., Moss, M.A. & Ellenberger, H.A. 1984, 'The cyanide content of laetrile
preparations, apricot, peach and apple seeds', Journal of Toxicology: Clinical
Toxicology, vol. 22, no. 4, pp. 341-7.
[8] Bradbury, J., Egan, S. & Lynch, M. 1991, 'Analysis of cyanide in cassava using acid
hydrolysis of cyanogenic glucosides', Journal of the Science of Food and Agriculture,
vol. 55, no. 2, pp. 277-90.