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Chemiphar Uganda Limited

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Traineeship proposition
Stageonderwerp 2012-2013: Validation of a method for the monitoring of Cadmium and Mercury in different food commodities
Stageonderwerp 2009-2010
Bepaling van de meetonzekerheid binnen de afdeling microbiologie
Elisa testen voor de screening van antibiotica
Stageonderwerp 2008-2009
Project: onderzoek van fumonisines in maïs, soja en unimix en deze in verband brengen met spina bifida.
Opdrachten: Meehelpen in routine, dit zijn de dagelijkse handelingen in een labo waar voedings- en milieuanalyses gebeuren.
Technieken: gravimetrie (wegen), vast-vloeistof extractie, vloeistofchromatografie, dunnelaag-
chromatografie, elisa, spectrofotometrie, TLC-plate spotter, TLC-scanner, microplaatreader, balans, …
Samenvatting eindwerk 2012-2013: Validation of a method for the monitoring of Cadmium and Mercury on different food commodities
This bachelorproject consisted two main parts: The analyzing of Heavy metals (Cadmium and Mercury) in different food samples. And the validation of the used methods.
Heavy metals occur naturally in the environment and they can be absorbed into the human body by a number of ways, which the intake of food is one of them. Cadmium(Cd) and Mercury(Hg) can cause toxic effects to the human health and that is why it is important that the food samples has been analyzed on heavy metals. The food samples that been analyzed are : Nile perch, Coffee beans, Cocoa beans and Unimix®.  The samples were prepared with a digestion technique. After the sample preparation, the samples were analyzed on Cd with the Flame-AAS(F-AAS) and Hg with the Flow injection mercury system(FIMS).
After analyzing the food samples on these heavy metals could be concluded that they were all negative on both heavy metals.
the validation of the used method is important because a validation can prove if a method is reliable or not. The validation was done on the basis of various parameters: limit of detection(LOD), limit of quantification(LOQ), linear range, repeatability, reproducibility and trueness. Because all food samples were negative on both heavy metals was it necessary to spike the samples otherwise the validation was impossible. For cadmium a spike was used of 25 mg/kg on a wet sample and one of 50 mg/kg on the dry sample. For Mercury a spike was chosen of 0,20 mg/kg on dry samples and of 0,10 mg/kg on wet samples.
The validation of the method that was used for Cadmium showed that the LOD is set on a concentration of 0,023 mg/kg and the LOQ is set on 0,05 mg/kg. The linear range is located between a respons factor of 0,1736 an 0,1919. Because the RSD %  of the Dry and wet samples respectively  4,67% and 1,80% is lower than the 2/3 H-value (6,70 and 5,96) is proven that the method is repeatable. And because of the same RSD% is lower than 4/3 H-value (13,40 and 11,92) is shown that the method is reproducible. Also the used Dixon Q-test didn’t detected any outliers. The parameter was controlled by recovery that should be within 80 and 110%. The results of the dry samples where 100%, 101% and 99% and the one for the wet samples where 97%, 101% and 101% with where within the parameters.
The validation of the method that was used for the determination of Hg gave a LOD of 0,000096 mg/kg and a LOQ of 0,00019 mg/kg. The linear Range is set between respons factor of 0,0282 and 0,0311. The Dixon Q-test showed that there were no outliers. The RSD% of the Dry sample (5,85%) and of the wet sample (13,26%) where lower than the 2/3 H-value (13,84 and 15,35) and 4/3 H-value( 27,68 and 50,69) So can be concluded that the method is repeatable and reproducible.  The results of the recovery for dry samples ( 104%, 94% and 95%) and of the wet sample( 105%, 103% and 97%) where between de set parameters.
So as final conclusion can be said that both methods fulfilled all set parameters.
Samenvatting eindwerk 1 2009-2010: Validation of 3 ELISA-kits for screening of antibiotics in honey – Determination of lead in fish with graphite furnace AAS
Honey is a product that is eaten over the whole world.  Although honey is healthy, consuming honey could bring some danger with. Sometimes, beekeepers put antibiotics in the apiculture to protect their bees from diseases and infections. This could be dangerous first of all for bacterial resistance and secondly it can cause damage to the human body when consuming the honey. Since 2005, Uganda is authorized to export honey to the EU as it meets European legislations concerning the import of honey. This means that the honey must be free of all substances that may cause danger to humanization. Nowadays, Chemiphar (U) Ltd. gets requests for determination of antibiotics in honey samples. Chloramphenicol, streptomycin and multi-sulphonamides are three antibiotics that are determined with a competitive Enzyme-Linked Immuno Sorbent Assay (ELISA). To prove that the results of the screening are true and that the method is applicable and acceptable for its intended purpose, it needs to be validated. Hereby, validation parameters such as repeatability, reproducibility, selectivity, ruggedness need to be determined and a detection capability CCβ has to be set.
The kit for determination of chloramphenicol and streptomycin is found to be applicable in the lab and the detection capability CCβ is set at respectively 0.2 µg/kg and 11.7 µg/kg. Both kits are also repeatable and reproducible for their intended purpose. Concerning the kit for determination of sulphonamides no colour development occurred within the test and the results were not present. This could be due to the environmental conditions of the laboratory so therefore more experiments need to be done at different environmental conditions such as temperature, light intensity, climate, etc.
Chemiphar (U) Ltd. also gets a lot of fish samples mainly coming from Lake Victoria. This fish may contain residues of contaminants for example heavy metals, which are dangerous for human health. As Uganda is authorized to export fish to Europe, residue analysis is necessary.
Lead is a heavy metal that is determined in fish with a Graphite Furnace Atomic Absorption Spectrophotometer (GFAAS). To be sure that the results that are claimed are true, the method must be validated. Next to that, the measurement uncertainty on the instrument is determined to give a good interpretation of the results.
One concluded that the expanded measurement uncertainty is found to be 67,2% for 0,1 mg/kg and 142,4% for 0,3 mg/kg. These uncertainties are too high especially due to high bias values. This could be caused by errors in the system itself or contamination during pretreatment of the sample. To check this, more experiments need to be done with different analysts and with better calibrations.  If these factors are improved, measurement uncertainty could be lowered and the method can be optimized.
Samenvatting eindwerk 2 2009-2010: The validation of three standard kits for the screening of antibiotics in food and determination of measurement uncertainty in microbiology
Honey is a delicious viscous sweetener made naturally by bees. Given its origin, honey has always had the image of a healthy nature product. No additives, preservatives or taste substances can be added. But just like other natural products honey can contain residues, miniscule traces of strange substance (e.g. antibiotics, pesticides, heavy metals and etc). The presence of antibiotic residues in honey is very problematic. A frequent exposure to low level antibiotics can cause resistance to micro-organisms. They can mutate, so the antibiotics are ineffective when needed to fight a human infection. When beekeepers want to export honey to the European Union, they have to make sure that the honey complies with all EU requirements in order to be allowed to the EU market. Around 1990 the European Union laid down the procedure for launching the maximum residue levels (MRL) of veterinary drugs used in food from animal origin. Since mid-2005, the Uganda honey has been certified to meet European Unions (EU's) legislated standard for honey import to European Union (EU) to Council Directive 96/23/EEC. The validation of the screening tests is necessary so that Chemiphar Uganda ltd can do the screening itself. In the past all samples needed to be shipped to Bruges for screening and quantification. In this project one tested two screening methods for the determination of antibiotics in honey (Premi®test and the Tetrasensor®).
The Premi®test (DSM food industries, Netherlands) is a multi-class inhibition test which gives a colour change. The test as such is not sensitive enough to meet with the minimum required performance level (MRPL). For this reason a selective extraction for tylosin was used to meet with the MRPL. The CCbeta of the test is determined on 20 µg.Kg-1.
The Tetrasensor® is a qualitative method, it’s a competitive test that exploits the activity of a receptor for the recognition of tetracycline molecules present in honey. The CCbeta of the test is determined on 20 µg.Kg-1. The method is specific to all tetracycline compounds. The test is easy to use and it is a quick and significant time saving operation.
The methods proved to be applicable to honey and also to be robust.
In this project also one screening method for the determination of beta-lactams in milk is validated. Milk has been a food source for humans since prehistory. The nutritional value of milk is very high. Antibiotics are used to control mastitis and other diseases in dairy cattle. The presence of antibiotic residues in milk is very problematic. The Council regulation (EEC) No 2377/90 of 26 June 1990 laying down a community procedure for the establishment of maximum residue limits of veterinary medicinal products in foodstuffs of animal origin. The milk production in Uganda has experienced steady growth, but it’s still low.
The Delvo®test is a qualitative method, it’s a microbiological inhibition test which gives a colour change. The Delvo®test is also a multi-class inhibition test (responds to several antibiotics). The test is an inexpensive, easy to perform screening test. The method proves to be applicable to milk. The screening method is not selective for one antibiotic, that means that one never know which one is present, unless conformation is done. The method proved also to be robust (same samples were analysed on different times).
A positive result of the screening methods needs to be confirmed by a selective quantitative method, more specific LC-MS/MS in Chemiphar at Bruges.
The last part of the project is to determine the measurement uncertainty for quantitative determinations in the microbiology. Different categories are tested. In each category the measurement uncertainty of each relevant micro-organism for the laboratory is determined, namely:
  • Staphylococcus aureus (category four);
  • Total coliforms (category three and four);
  • Escherichia coli (category three and four);
  • Yeast and moulds (category four).
The measurement uncertainty is necessary to take into account the uncertainty associated with the result to see of the obtained value meets the European guidelines.
Samenvatting eindwerk 2008-2009: Determination of fumonisins in corn and unimix with ELISA and TLC-densitometry
Fumonisins are mycotoxins produced by moulds. They can be found in food long after the moulds have died. They are known to be toxic, and are a cause for spina bifida. In Uganda a lot of soybeans, corn and unimix is stored and consumed. As a result we would which to have an idea of the fumonisin content in food supplies. For this 2 methods are used, a fast screening with ELISA and a confirmation method with TLC detection.
When testing three TLC methods from literature, none of these methods worked well. A first remark is that the corn samples interference with the fumonisins, so the Rf measurement was different as in the literature. Therefore a cleanup and extraction step with SPE step was needed. A second remark is that the coloring reaction did not worked well on the fumonisins, so another coloring solution as drying condition is used after testing. After optimization, the TLC technique is not very sensitive because it only starts measuring fumonisins at a concentration of 20ppm. Samples have to be spiked to get a measurement, so this technique is a semi-quantitative technique. The recovery percentage is lower as 60%, this can be caused of the changed SPE method.
The ELISA is a RIDASCREEN fumonisin test which is a competitive enzyme immunoassay for the quantitative analysis of fumonisinB1, B2 and B3 in corn and corn products. It has a sensitivity of 0.025ppm. When validating the test, the results were not so good. The standard deviation was quite high after a repeatability and reproducibility study.
TLC is a promising technique for the detection of fumonisins, although it is semi-quantitative. More experiments to get a better dynamic range are needed e.g. HPTLC can be used instead of TLC. A better way has to be found for getting a better concentration of solvent into the matrix with SPE. The more expensive ELISA has a better sensitivity and should be better as TLC.


Acacia Road
(256-41) 26 88 32


Traineeship supervisor
Jan Cordonnier
Traineeship supervisor
Van Quekelberghe Stijn
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