Development and Validation of a Bioanalytical method for the Determination of Levamisole Residue in Backyard Poultry Egg

 

Md Kamal Hossain¹*, Kamrun Nahar¹, Mohammed Ehsanul Hoque Mazumder¹,

Md Wahiduzzaman¹, Ali S. Alqahtani², Dr Tony Gestier¹, Kaiser Hamid¹*

¹Vetafarm Pty Ltd. R and D Centre, Wagga Wagga, NSW, 2650, Australia.

²Faculty of Pharmacy, King Saud University, Saudi Arabia

 

ABSTRACT:

Development of a bioanalytical method is often tricky and labour intensive due to its nature or the criteria set by regulatory bodies. In the present study, a simple, reliable and sensitive analytical method was developed for the determination of levamisole residue in chicken eggs. Levamisole and ronidazole (internal standard, IS) were separated on a C₁₈ reversed-phase HPLC column. Following liquid-liquid extraction, chromatographic separation was accomplished with a mobile phase consisting of acetonitrile: 0.05M KH₂PO₄ at ratio of 20:80, and the drug was detected at 235nm using a UV detector at a flow rate of 1.0ml/min at the ambient temperature. Linearity was obtained over the range 0.3125–10.0μg/ml for levamisole hydrochloride with lower limit of quantitation of 0.3125μg/ml. For each level of quality control samples, inter- and intra-day precision (%CV) was within ±15%. Absolutes extraction recovery of drug from plasma was ≥60%.The residue limit of levamisole gradually increased from day 1 to day 3 and then declined from day4 to day 7. The highest level of residue after oral administration of 80mg/kg levamisole to chickens was on day 3 (0.599μg/g). This level was lower than the recommended maximum residue limit (MRL), thus, no withholding period was required for egg consumption following the administration of the levamisole solution.

 

 

 

 

1.1 INTRODUCTION:

The requirement of veterinary drugs or veterinary medicinal products (VMPs) are inevitable to meet the challenges of providing adequate amounts of food for the growing world population¹as these drugs improve the rate of weight gain, improve feed efficiency, or prevent and treat diseases in food producing animals^1,2. However, the benefit of improved productivity from the use of these drugs in food producing animals is not risk free as the residues that remain in the tissues of treated animals at the time of slaughter ^1,3 or residues in animal derived products and poses a health hazard to the consumer.

 

 

Globally, there is a growing concern about residues in meat and eggs and it is likely that the requirements for testing for residues will be increased in the future. According to the Food and Marketing Institute Report (1988), food safety has become one of the most critical factors confronting affluent societies⁴.

 

However, the detection/identification of veterinary pharmaceuticals residues from food sources has always been considered a challenging practice for an analytical/R and D chemist.

 

 

Figure 1. Chemical structure of LMS

 

Levamisole (LMS) is (S)-6-Phenyl-2,3,5,6-tetrahydroimidazo[2,1-b][1,3]thiazole (Figure 1), a pharmaceutical product with anthelminthic and immunomodulatory properties that have been previously used in both animals and humans to treat inflammatory conditions and cancer⁵. LMS is widely used as an anthelmintic in cattle, sheep, goats, swine and poultry. It has been used in backyard poultry for the management of internal parasites, especially for roundworm, and is currently being used to treat capillaria infections in poultry⁶. It is effective against lungworms and gastrointestinal nematodes. It is also used as an adjuvant therapy in the treatment of human cancer⁷.

 

In a national survey, it was found that the first concern of consumers pertains to residues in meat. The recommendations for doses of LMS to be used in poultry are available⁸, but these are not approved for use in the USA. LMS is approved in Australia and is administered at a dose of 28mg/kg⁹ . A substantial literature survey revealed a lack of information from the Food Animal Residue Avoidance Databank (FARAD) and the UN Food and Agriculture Organization (FAO) regarding the residue potential, metabolism or withdrawal times of LMS used in poultry¹⁰. In the USA, the maximum residue limit (MRL) in edible tissues is 0.1μg/g, which only applies to cattle, sheep, goats and swine⁶,¹¹. The withdrawal time is different in various animal species and it is hard to predict the withdrawal time in chickens after they are medicated with LMS. The concentrations of LMS could be different based on different formulations and eventually this could affect the withholding period as well. As such, it is crucial to determine the residue limit of the newly developed LMS formulation intended for the backyard poultry industry.

 

There are few analytical methods that reported the determination of LMS in bulk and pharmaceutical preparations and in biological fluids by using spectrophotometry¹², HPLC (High Performance Liquid Chromatography)^13-18 and LC-MS^19-20 techniques along with just one study for the determination of LMS residue in chicken tissue and eggs which involve the complex extraction and analytical process ²¹. It is also noteworthy to mention that the internal standards used in the above article are not easily accessible. Additionally, most of the available methods have limitations such as long runtimes, low sensitivity, uneconomical, poor symmetry and often involve very complicated extraction processes which has triggered the necessity for developing a simple, sensitive and user-friendly analytical method. Keeping this in mind, this study was conducted to develop and validate a simple, accurate, precise and reliable RP-HPLC method for the detection and quantification of LMS residue in backyard poultry eggs following the administration of LMS oral solution (Laying Hen Worm Out^®) at the recommended dose.

 

1.2. MATERIALS AND METHODS:

1.2.1. Materials:

LMS HCl and ronidazole were purchased from Sigma Aldrich(Castle Hill, Australia).All other chemicals of analytical grade were purchased from commercial sources(Merck Millipore, USA and Chem Supply, Gillman, South Australia). Water was purified using the Ultrapure Water System (Sartorius). All solvents, including water, used for extraction and in the mobile phase were of HPLC grade purity and used without further purification.

 

1.2.2. Apparatus and chromatographic condition:

The HPLC system (2000 series, Shimadzu) consisted of a pump with a column from Gemini C₁₈ (4.6mm × 250mm), and a UV-detector with a data processor (Lab Solutions software) was employed. UV detection for LMS and ronidazole was set at 235nm. The mobile phase consists of 0.05 M KH₂PO₄: acetonitrile (80:20) delivered at a flow rate of 1.0ml/min at the ambient temperature. Peak identities were confirmed by retention time (RT) of LMS HCl at 7.5 minutes and ronidazole (internal standard) at 8.2 minutes. A Labofuge 200(Thermo Scientific, USA) was used for centrifuging the egg sample and Reactitherm Module III(Thermo Scientific, USA) was used to dry the supernatant under a stream of N₂ gas.

 

1.2.3. Preparation of stock solutions and quality control samples:

The standard stock solution of LMS and ronidazole (1.0mg/mL) was prepared in methanol. The working standard solution was prepared by diluting the stock solution in blank egg (Table 1).

 

Table 1: Preparation of working standards from stock solution

 

 

 

1.2.4. Extraction process:

A simple liquid–liquid extraction process was employed where 1g whole egg (eggs were homogenised by hand mixer at medium speed) were homogenised by adding 0.5mL of water and 0.5mL of acetonitrile for 3 minutes. After adding 100µl of 1N NaOH and 2 mL of ethyl acetate, the mixture was shaken for 3 minutes followed by centrifugation for 5 minutes at 5000 rpm. The supernatant was collected and dried with stream of liquid N₂ gas and after filtering with NY 0.45µm syringe filter, the solution was injected into the HPLC.

 

1.2.4. Linearity:

A 1mg/ml stock solution was prepared by dissolving 50mg LMS HCl reference standard (Sigma) in 50ml of methanol. The working standard concentrations for LMS HCl were 0.3125, 0.625, 1.25, 2.5,5 and 10µg/mL which were prepared by diluting the stock solution with blank egg. Ronidazole (20µL) was added to each concentration point after the dilution with blank egg. The LMS HCl(20µl)stock solution (1mg/mL) was mixed with 1.980g blank egg creating the 10µg/g working standard. Then 1ml of this solution was mixed with 1mlof blank egg, becoming 5µg/g. The rest of the working standards were prepared following the same calculation technique (Table 1).

 

1.2.5. Specificity;

The specificity of the method was established using different concentrations of LMS. In each batch of analysis, blank samples and the LLOQ (lower limit of quantitation) were processed and analysed with an assay procedure to determine any significant interference with the RT of the analyte.

 

1.2.6. System suitability:

A system suitability test was used to verify that the resolution and reproducibility of the chromatographic systems were adequate for the analysis to be accomplished. The tests were based on the fact that the equipment, electronics and samples to be analysed constituted an integral system that could be evaluated as such. The limits for system suitability were set for theoretical plates, tailing factor and RT.

 

1.2.7. Extraction recovery:

Recovery of the LMS HCl and ronidazole was evaluated by comparing the mean peak areas of LQC, MQC and HQC quality control samples with the mean peak areas of six reference solutions containing the same amount of the test compound.

 

1.2.8. Precision and accuracy:

Intra- and inter-day precision and accuracy of the developed method were evaluated in plasma samples spiked with LMS HCl at concentrations of 0.3125, 2.5 and 10 μg/ml and assayed. Inter-day precision and accuracy were evaluated on two consecutive days. The criteria for acceptability of the data included accuracy within ±15% of the coefficient of variation (CV) from the nominal values and a precision of within ±15% of the CV, except for LLOQ, where it should not exceed ±20% for accuracy and precision.

 

1.2.9. Stock solution stability:

The working solution (1.0mg/ml) of LMS HCl was repeatedly (n=3) injected for analysis, immediately after preparation (0 hours) and at 3, 6 and 9 hours after bench top storage at room temperature and at 4°C. This injection protocol was repeated after 15 and 30 days’ storage of the stock solution between 4–8°C.

 

1.2.10. Dilution integrity:

A dilution integrity experiment was performed with the aim to validate the dilution test to be carried out on a higher analyte concentration above the upper limit of quantification (ULOQ), which maybe encountered during real subject sample analysis. Six replicates each, at a concentration of double the uppermost calibration standard, were diluted two-fold and eight-fold with blank plasma. The diluted samples were processed and analysed against the calibration curve.

 

1.2.3. Experimental design for egg residue study:

The egg residue study was conducted according to Vetafarm R and D Centre’s animal handling guidelines. Fifty Light Sussex chickens were hatched and raised in a free-range situation on a private property until commencing egg laying. The chickens were raised on a drug-free grain mix with adlib intake. Water was provided from the reticulated, potable town water supply. Once laying patterns had been established, two groups of 6 chickens each were separated into holding pens equipped with nest boxes. Hens were allowed to accommodate to the changed environment to ensure the regular laying of eggs.

 

The test group of chickens (n=6) were administered a single dose of Laying Hen Worm Out^® Solution at the rate of 80mg/kg orally (Table 2). Each chicken was weighed using a set of digital scales (5kg) with 2g increments and their weights recorded. The eggs from the treatment and control groups were collected from day 1 to day 7 and were refrigerated until the analysis was performed.

 

Table 2: Calculation of LMS administered to chickens

Chicken body weight(kg)	Amount of Worm Out solution (ml)
2.70	13.50
2.70	13.50
2.90	14.50
2.70	13.50
2.87	14.35
2.96	14.96

 

1.4. RESULTS:

1.4.1. Selectivity:

Blank egg sample from 3 different sources was injected after necessary treatment to HPLC. No interference peak was observed at the RT of LMS HCl (7.524 minutes) or ronidazole (8.194 minutes) (Figure 2).

 

Figure 2. Sample chromatogram: A: Blank egg, B: Egg sample with LMS

 

1.4.2. Carry-over:

Carry-over in the blank sample following the high concentration of standard should not be greater than 20% of LLOQ and 5% for the internal standard(EMEA). Blank egg samples were injected following ULOQ during intra-day validations. In the present study, the carry–over limit for LMS HCl was 8.22% and for ronidazole, it was 1.28%.

 

1.4.3. Linearity:

Calibration was found to be linear over the range of 10 to 0.3125 µg/ml of LMS HCl with a very good correlation coefficient of 0.9985(Figure3).

 

Figure 3. Linearity curve for LMS HCl over the range 0.3125 to 10 µg/ml

1.4.4. Within-day Precision and Accuracy:

The mean concentration was within 15% of the nominal values for the QC samples, including the LLOQ and precision (%CV), which was also within the specifications (Tables 3 and 4).

 

Table 3: Within-day validation (concentration, µg/ml)

	Within-day validation (concentration, µg/ml)
Concentration (µg/ml)	0.31	0.63	2.50	10.00
1	0.28	0.72	2.75	9.50
2	0.31	0.66	2.82	9.37
3	0.29	0.68	2.80	9.56
4	0.27	0.62	2.75	9.46
5	0.29	0.69	2.82	9.40
6	0.29	0.71	2.80	9.40
Mean	0.29	0.68	2.79	9.45
SD	0.012	0.037	0.033	0.075
CV	4.134	5.451	1.199	0.795

 

Table 4: Within-day validation (accuracy %)

	Within-day validation (accuracy %)
Concentration (µg/ml)	0.31	0.63	2.50	10.00
1	89.2	97.3	109.9	95.0
2	98.7	101.3	112.9	93.7
3	92.9	97.9	111.9	95.6
4	87.8	98.4	109.9	94.6
5	93.0	102.6	112.9	94.0
6	93.7	105.5	111.9	94.0
Mean	92.54	100.50	111.56	94.50
SD	3.82	3.21	1.35	0.81
CV	4.10	3.21	1.23	0.86

 

1.4.5. Between-day Precision and Accuracy:

The mean concentration was within 15% of the nominal values for the QC samples, including the LLOQ and precision (%CV), which was also within the specifications (Tables 5 and 6).

 

Table 5: Between-day validation (concentration, µg/ml)

Concentration (µg/ml)	Between-day validation (concentration, µg/ml)
	0.31	0.63	2.50	10.00
1	0.37	0.71	2.43	9.16
2	0.37	0.66	2.53	9.69
3	0.35	0.69	2.45	9.00
4	0.36	0.59	2.54	9.20
5	0.37	0.71	2.46	9.55
6	0.34	0.71	2.57	10.28
Mean	0.36	0.68	2.50	9.48
SD	0.013	0.048	0.056	0.469
CV	3.505	7.128	2.240	4.945

 

Table 6:Between-day validation (accuracy %)

Concentration (µg/ml)	Between-day validation (accuracy %)
	0.31	0.63	2.50	10.00
1	99.1	113.3	105.5	114.3
2	90.5	105.4	111.7	113.5
3	97.9	110.2	86.4	92.7
4	94.5	93.7	101.5	108.0
5	99.5	113.1	100.0	95.1
6	96.40	113.20	104.00	113.61
Mean	96.30	108.14	101.53	106.20
SD	3.42	7.71	8.42	9.84
CV	3.52	7.13	8.34	9.31

1.4.6. Recovery study:

Liquid-liquid extraction was employed for the extraction of the drug and the internal standard from the egg sample. In the current extraction method, there was more than 60% recovery for LMS and ronidazole (Table7).

 

Table 7:Extraction recovery study data

% Recovery
Concentration (µg/ml)	0.31	0.63	2.50	10.00	Ronidazole
1	81.088	62.100	55.521	62.883	64.699
2	79.818	57.085	50.263	66.033	64.385
3	81.669	52.990	54.096	62.664	65.507
4	79.615	57.053	57.895	61.734	63.224
5	85.067	54.304	56.015	66.033	65.320
6	80.164	56.989	61.899	69.558	64.129
Mean	81.237	56.753	55.948	64.817	64.544
SD	2.034	3.131	3.876	2.943	0.836
CV	2.503	5.517	6.929	4.541	1.295

 

1.4.7. Dilution integrity:

Accuracy and precision of final concentration was within ±15%. In the present study, the dilution of samples did not have any effect on the accuracy or precision of analysis (Tables 8 and 9).

 

Table 8: Egg sample subject to 8x dilution

Diluted conc. (µg/ml)		Peak area of LMS	Peak area of ronidazole	Peak ratio	Measured conc. (µg/ml)	Accuracy (%)
2.5	A	29231	76470	0.2776	2.43	97.27
	B	26463	78249	0.2871	2.53	101.30
	C	28481	75695	0.2790	2.45	97.87
	D	30481	78222	0.2874	2.54	101.44
	E	29491	76660	0.2803	2.46	98.43
	F	32589	77841	0.2902	2.57	102.63
				Mean	2.50	99.82
				Std Dev	0.06	2.24
				CV, %	2.24	2.24

 

Table 9:Egg sample subject to 2x dilution

Diluted conc. (µg/ml)		Peak area of LMS	Peak area of ronidazole	Peak ratio	Measured conc. (µg/ml)	Accuracy (%)
10	A	109454	120599	0.9076	9.16	91.57
	B	114937	120014	0.9577	9.69	96.92
	C	109073	122104	0.8933	9.00	90.05
	D	107454	117850	0.9118	9.20	92.02
	E	114937	121756	0.9440	9.55	95.46
	F	121073	119536	1.0129	10.28	102.81
				Mean	9.48	94.81
				Std Dev	0.47	4.69
				CV, %	4.94	4.94

 

Table 10:Concentration of LMS (µg/g) in chicken eggs (mean ± SD) after oral administration of 80mg/kg LMS to chickens (n=3)

Sample	Day 1	Day 2	Day 3	Day 4	Day 5	Day6	Day 7
Egg	0.188±0.96	0.487±1.25	0.599±1.55	0.306±0.75	0.129±0.75	0.090±0.98	0.096±1.29

 

1.5. DISCUSSION:

The concentration of LMS in egg samples after oral administration of 80mg/kg is summarised in Table 10. The results demonstrated that the LMS concentration increased gradually from day 1 to day 3 and declined from day4 to day7 after administration. The results from this study indicates that the highest residue concentration of LMS was found on day 3 which was less (0.599µg/g) than the maximum residue limit (1μg/g) for eggs. The residue result trend in this study is consistent with the study conducted by El-Kholy and Kemppainen (2005)²¹where on day 3, the LMS concentrations was 0.55µg/g. Although there is no published information about the withdrawal time of LMS in chickens in the USA but  in Australia, the withdrawal times for chicken tissue and eggs are 7 and 0 days respectively ²²and the maximum residue limit of LMS in chicken tissue and eggs are 0.1 and 1μg/g respectively²¹(El-Kholy and Kemppainen, 2005).The residue limit of LMS for the current worm out solution (Laying Hen Worm Out^®)is well below (0.599µg/g) of the Australian requirement, thus does not require any withholding period for eggs.

 

The new HPLC assay method presented herein for the assay of LMS HCl residue in chicken eggs using a C₁₈ reverse phase column was found to be valid for the Vetafarm product Laying Hen Worm Out^® solution. The method was linear over the concentration ranges of 10 to 0.3125 µg/ml) of LMS HCl with a correlation coefficient of 0.9992. In this study, the carry–over limit for LMS HCl was 8.22% and for ronidazole, 1.28%.The mean concentration was within 15% of the nominal values for the QC samples, including the LLOQ and precision (%CV), which were also within the specifications. Dilution of samples by 2x and 8x did not affect the accuracy or precision. This method, thus, offers a simple extraction and assay method for the determination of LMS residue in eggs and could be an attractive alternative to the existing method.

 

1.6. CONCLUSION:

The new HPLC assay method described herein for the assay of levamisole HCl residue in chicken egg using a C₁₈ reverse phase column was found to be valid for the Vetafarm product Laying Hen WO^(R) solution. The method was linear over a concentration range examined (10 to 0.3125 µg/ml) of levamisole HCl with a correlation coefficient of 0.9992.  In this study, the carry –over limit for levamisole HCl was obtained 8.22% and for ronidazole, it was 1.28 %. The mean concentration was within 15% of the nominal values for the QC samples, including the LLOQ and Precision (% CV), which were also within the specifications. Dilution of samples by 2x and 8x did not affect the accuracy and precision. This method, thus, offers a simple extraction and assay method for the determination of levamisole residue in egg and could be an attractive alternative to the existing method.

 

1.7. CONFLICT OF INTEREST:

Md Kamal Hossain, Mohammed Ehsanul Hoque Mazumder, Md Wahiduzzaman, Kamrun Nahar, Ali S. Alqahtani, Dr. Tony Gestier, and Kaiser Hamid declared that they have no conflicts of interest.

 

1.8. REFERENCES:

1.       Crawford LM: The impact of residues on animal food products and Human Health. Rev Sci tech Off Int Epiz 1985; 4: 669-685.

2.       Antimicrobial use and antimicrobial resistance: American Veterinary Medicine Association (AVMA), 2015.

3.       Health Canada: Maximum residue limits for veterinary drugs, 2013.

4.       O’flynn M: The NRS and Australia's management of chemical residues. 17^th Conference of Residue Chemists, 1999; pp. 24-26

5.       Ravisankar P, Rao GD: Development and validation of RP-HPLC method for determination of Levamisole in bulk and dosage form. Asian. J .Pharm. Clin. Res. 2013; 6: 69-173.

6.       USDA National Residue Program Plan, Food Safety Inspection Services. Office of Public Health and Science, Washington, DC 1998.

7.       Gwilt P, Tempero M, Kremer A, Connolly M and Ding C: Pharmacokinetics of levamisole in cancer patients treated with 5-fluorouracil. Cancer chemo. and pharmacol. 2000; 45:247-251.

8.       Plumb C: Veterinary Drug Handbook., Iowa State University Press, Ames, IA, 1999.

9.       Arundel JH: Veterinary Anthelmintics. University of Sydney, Postgraduate Foundation in Veterinary Science, New South Wales, Australia, 1985.

10.     Sundlof SF, Riviere JE and Craigmill AL: FARAD—The food animal residue avoidance databank., University of Florida, Gainesville, FL, 1992.

11.     Craigmill AL, Sundlof SF, Riviere JE: Hand Book of Comparative Pharmacokinetics and Residues of Veterinary Therapeutic Drugs. CRC Press, Boca Raton, FL, 1994.

12.     El-Didamony AM: Spectrophotometric determination of benzydamine HCl, levamisole HCl and mebeverine HCl through ion-pair complex formation with methyl orange. Spectrochimica Acta Part A: Mol. Biomol. Spectroscop. 2008; 69: 770-775.

13.     Garcia J, Diez M, Sierra, M and Terán M: Determination of levamisole by HPLC in plasma samples in the presence of heparin and pentobarbital. J. Liq. Chromatogr. 1990;13,743-749.

14.     De Bukanski BW, Degroodt JM, Beernaert H: Determination of levamisole and thiabendazole in meat by HPLC and photodiode array detection. Eur Food. Res. Technol. 1991; 193: 545-547.

15.     Marriner S, Galbraith E and Bogan J: Determination of the anthelmintic levamisole in plasma and gastro-intestinal fluids by high-performance liquid chromatography. Analyst, 1980; 105, 993-996.

16.     Du Preez J, Lotter A: Solid-phase extraction and HPLC determination of levamisole hydrochloride in sheep plasma, 1996. 

17.     Sari  P, Sun J, Razzak M and Tucker IG: HPLC assay of levamisole and abamectin in sheep plasma for application to pharmacokinetic studies. Journal of liquid chromatography  and  related technologies. 2006; 29: 2277-2290.

18.     Swartz ME: UPLC™: an introduction and review. J. liq. chromatogr.  reltech. 2005; 28, 1253-1263

19.     Cannavan A, Blanchflower W, Kennedy D: Determination of levamisole in animal tissues using liquid chromatography–thermospray mass spectrometry. Analyst. 1995;120: 31-333.

20.     Chappell CG, Creaser CS, Stygall JW, Shepherd MJ: On‐line high‐performance liquid chromatographic/gas chromatographic/tandem ion trap mass spectrometric determination of levamisole in milk. Bio. Mass. Spectrom. 1992;21: 688-692.

21.     El-Kholy H, Kemppainen B: Levamisole residues in chicken tissues and eggs. Poultry sci. 2005; 84, 9-13.

22.     Health CA:Coopers Animal Health. http://www.coopersanimalheath.com.au/, Coopers Animal Health, Baulkham Hills, NSW, Australia, 2004.

 

 

 

 

 

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