Validation of a Method for Analysis of Sulfa Drugs in Poultry and Beef Samples using Liquid Chromatography Tandem Mass Spectrometry

doi:10.21203/rs.3.rs-1958777/v1

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Validation of a Method for Analysis of Sulfa Drugs in Poultry and Beef Samples using Liquid Chromatography Tandem Mass Spectrometry

https://doi.org/10.21203/rs.3.rs-1958777/v1

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Antibiotics residue of any drug above the allowable limit in poultry and cattle farms reduces food safety and quality. The main aim of this study was to develop and validate a sensitive and reproducible method to assess the amount of residual limit of antibiotics. Poultry meat and beef samples were analyzed for the presence of six sulfa drugs i.e., sulfathiazole (STZ), sulfadiazine (SDZ), sulfamerazine (SMZ), sulfamethazine (SMT), sulfamethoxypyridazine (SMP) and sulfadimethoxine (SMX) by LC-MS/MS coupled with ESI and QQQ mass analyzer. Identification and quantification of drugs were carried out by SCAN and MRM methods. Matrix matched calibration curves were linear with correlation coefficients (r²) 0.997, 0.998, 0.997, 0.993, 0.996 and 0.998 for six drugs, respectively. The method was validated by recovery experiments at 2 spiking levels (5 & 10 ng/mL). Extraction was done by QuEChERS method, cleaned up was carried out with C-18 powder & PSA followed by analysis using LC-MS/MS. Intra- and inter-day experiments were done with desirable accuracy and precision, and recoveries were in the range of 86-104%. Poultry meat (n=30) and beef samples (n=30) were analyzed from thirty different markets, super shops, and residues of six sulfa drugs were found below the detection limit.

Antibiotics

beef samples

LC-MS/MS

poultry meat

sulfa drugs

Antibiotics are used as growth promoter to enhance production and as medicine for the treatment of sick animals and as an antidote for the prevention of infection in poultry farm animals¹. Sulfa drugs are common antibiotics that are most widely used in veterinary medicine practice in livestock and poultry farms to combat many common bacterial disease treatments because of their low cost, relative efficiency and wide-spectrum antimicrobial activity^2,3. Sulfathiazole (STZ), sulfadiazine (SDZ), sulfamerazine (SMZ), sulfamethazine (SMT), sulfamethoxypyridazine (SMP) and sulfadimethoxine (SMX) are permitted for use as a veterinary drug in poultry feed⁴. Sulfa drug can be used to treat bacterial infections caused by Salmonella species, Clostridium species, Mycoplasma species etc⁵. However, poor animal husbandry with inappropriate use of these drugs residue may accumulate in edible tissues and can lead to the development of resistant bacteria or to the contamination of livestock products with sulfa drugs residue^6,7. When animals have been fed antibiotics over a period of time, bacteria retain the strains of drug resistance⁸. Eating meat from affected animals may lead to various potential hazards such as allergic reactions, bacterial resistance, toxic reactions, and teratogenic and carcinogenic effects on the human body^9,10. Many countries in the world have set up monitoring programs for observing drug residue levels in different sources¹¹. According to the European Union (EU) maximum residue limit (MRL) for sulfa drugs is 100 µg/kg in food of animal origins such as meat, milk, and egg¹².

Many analytical methods have been developed including TLC¹³, UV-Visible spectrophotometry¹⁴, gas chromatography (GC), capillary electrophoresis, and high-performance liquid chromatography (HPLC)¹⁵ were used for the detection of sulfa drugs residue levels. Because of suffering from low precision issues capillary electrophoresis method is not used widely for routine analysis. In the case of the development of methods based on GC and gas chromatography-mass spectrometry (GC-MS), polar sulfa drugs are required to be chemically derivatized to decrease their polarity and increase their volatility¹⁶. HPLC with photodiode array (PDA) detection has been applied to determine several sulfa drugs residue in meat, egg, and milk¹⁷. Chromatographic methods based only on ultraviolet or PDA detection are not the proper confirmatory methods as the matrix interference from higher concentration additives and lipophilic compounds in feedstuffs is a serious hindrance to the accurate quantitation of sulfa drugs residue in feedstuffs. The extractions of sulfa drugs from meat and milk were carried out using solid-phase extraction (SPE), liquid-liquid extraction (LLE) and matrix solid-phase dispersion (MSPD)¹⁸. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is the most widely used method for quantification and confirmation of drug residues in foods in recent years. High selectivity and sensitivity are obtained from LC-MS/MS when compared with all the methods mentioned above^19–26. LC-MS/MS method is used for the analysis of sulfa drugs in foodstuffs, such as tissues, eggs²⁷, milk²⁸, and honey²⁹.

A field survey was conducted on different farms and shops of poultry, and it was found that different antibiotics are being used in many poultry farms to treat infectious diseases in Bangladesh. Excessive use of antibiotics in food-producing animals is harmful to these animals and to humans which enters through the food chain. However, food safety and quality are very important for food consumption. Therefore, the aim of the present study is to develop and validate a method for analysis of sulfa drugs residue in poultry meat and beef samples by LC-MS/MS and the main objective of the extraction procedure is to get more accurate results for the analysis of a wide range of antibiotics.

Sample Collection

Poultry meat (n=30) and beef samples (n=30) were purchased from thirty different local markets and supermarkets in Dhaka city. Antibiotic-free chickens were collected from Muktagacha, Mymensingh, and used as a control. The samples were homogenized by using a kitchen blender and 10 g of each of the samples was taken in a screw cap Teflon tube (50 mL) and stored at -20 ⁰C.

Chemicals and reagents

Certified standards i.e., sulfathiazole (STZ), sulfadiazine (SDZ), sulfamerazine (SMZ), sulfamethazine (SMT), sulfamethoxypyridazine (SMP) and sulfamethoxine (SMX) were purchased from Sigma-Aldrich, primary secondary amine (PSA) from Supelco, USA, C18 powder were bought from Agilent Technologies, USA and LC-MS/MS grade methanol (MeOH), acetonitrile (ACN), magnesium sulfate (MgSO₄) and sodium chloride (NaCl) were also purchased from Sigma-Aldrich.

LC-MS/MS instrumentation

Liquid chromatography-mass spectrometry analyses were carried out using LC-MS/MS having electrospray ionization (ESI) and a triple quadrupole mass analyzer. Shimadzu Prominence ultra-fast liquid chromatography (column oven CTO-10AC; autosampler SIL-20ACHT) was used in this study. Separations were performed on a shim-pack GISS C18 column (250 × 4.6 mm i.d.; particle size 5 µm). N₂ gas was used as drying and nebulizing gas. Air and Ar gas were used as heating and collision gas, respectively.

LC Conditions

Separations were carried out in C18 reversed-phase column. Column oven temperature and flow rate of the mobile phase were set at 40⁰C and 1.0 mL/min, respectively. Extracted samples were injected through an autosampler and the injection volume was 20 µL. Mobile phases were a binary gradient elution of MeOH: H₂O with 0.1% formic acid (40:60 ratio).

MS/MS Conditions

The analysis mode was positive and the ionization process was electrospray ionization. Nebulising, drying, and heating gas flow rates were set at 3, 10, and 10 L/min, and interface, de-solvation gas line (DL), and heat block temperatures were set at 300, 250, and 400⁰C, respectively.

Standard Preparation

Stock solutions (1000 mg/L) were prepared first by taking 0.01 g of each sulfa drug in a volumetric flask (10 mL) with LC grade MeOH and finally from this stock solution, 10 mg/L primary standard solutions were prepared by 100 times dilution with MeOH. 100, 50, 25, 20, 15, 10, 5, 1.25, and 1.0 ng/mL working standard solutions were prepared from the primary standard solution using a mixture of MeOH-H₂O (80:20) as diluent.

Extraction and clean-up procedure

Homogenized samples were extracted by the Quick, Easy, Cheap, Effective, Rugged, and Safe (QuEChERS) method. In brief, the sample was taken into a teflon tube (50mL), then extracted using H₂O-ACN (20 mL; 1:1 ratio) followed by vortexed for about 3 min until homogeneity. Then a mixture of MgSO₄ (4.0 g) and NaCl (1.0 g) was added to the extract and again vortexed for 2 min followed by centrifugation (10 min) at 4000 rpm. The Upper ACN layer containing sulfa drugs were collected in another teflon tube and cleaned up was carried out using a mixture of PSA (0.15 g) and C18 (0.25 g). The cleaned extract was centrifuged (4 min) and a clear supernatant solution was passed through filter (0.22 µm), collected, and diluted with a mixture of MeOH: H₂O (4:1 ratio) for LC-MS/MS analysis.

Method Optimization

Analyses were first carried out using the flow injection method (FIM) in SCAN mode of Q1 and Q3 mass analyzers. Later, separations were performed by passing standard and sample through the LC column. The retention time of six sulfa drugs standards were 2.41, 2.42, 2.91, 3.59, 3.61, and 6.18 min for STZ, SDZ, SMZ, SMT, SMP, and SMX, respectively. Using collision gas in Q2 mass analyzers, three product ions were obtained listed in Table 1. Multiple Reactions Monitoring (MRM) modes were used for the detection of product ions. Precursor ions and 3 product ions were obtained in MRM optimization method and one product ion (highest intensity) was used for quantitation. Calibration curves were made with product ion of all the sulfa drugs.

Method validation

A method was validated in terms of linearity, accuracy, precision (repeatability and reproducibility), and sensitivity which are quantitative confirmatory parameters according to the EU Commission Decision, 2002/657/EC (European Commission, 2002)³⁰. Linearity was evaluated using matrix-matched calibration curves at six different concentration levels between 1.25-20 ng/g Table 2. For quantitation, the matrix-matched calibration curves of the respective drugs were used for effective quantification in meat samples. Poultry meat and beef samples were spiked with standards at 5 and 10 ng/g levels for the determination of accuracy and precision. Repeatability (intra-day) and reproducibility (inter-day) of the method were carried out by spiking meat samples at 5 and 10 ng/g levels as shown in Table 3. The precision of the method was estimated by determining the co-efficient variations calculated from results generated under the intra- and inter-day experiments and was obtained during the same day (n=5) and on three different days by the repeated analysis of a spiked sample. The limit of quantification (LOQ) and limit of detection (LOD) shown in Table 2 were estimated using the lowest concentration of sulfa drugs matrix-matched standard solution. This solution was analyzed three times and then on the basis of the statistical method, the obtained concentration was multiplied by 3 for LOD and 10 for LOQ³¹. The chromatogram for each sulfa drug standard is shown in Fig. 1.

Matrix effect

LC-MS/MS is selective and sensitive but often it may suffer from matrix effects. Method validation parameters may be affected by the matrix effect. The result of a sample was considered acceptable if its matrix effect was ≤85%³². So, the Matrix effect for each sample (%) was calculated with reference to the peak area of control sample and the peak area of standard.

Method optimization

Mixture of sulfa drugs standard solution was used for the optimization of both chromatographic and MS/MS parameters in this study. Chromatographic conditions were optimized for better quantification of sulfa drugs in meat samples which involved optimization of mobile phase composition, injection volume, flow rate, and column oven temperature. For symmetrical elution and reproducible retention time of each of the product ion, several mobile phases with varied concentrations were used, and MeOH: H₂O (40:60) ratio gives the best elution and high sensitivity. The optimized column oven (40˚C) temperature, injection volume (20 μL), and shim- pack GISS C18 column produced the best result in analysis of sulfa drugs residue. Electrospray ionization used as ion source for the identification and quantification of sulfa drug, gave good linearity of calibration curve. The precursor ion of each sulfa drug shown in Table 1 was first optimized by injecting a 250 ng/mL standard solution of sulfa drug in both positive and negative polarity mode. Formic acid was used in mobile phase and due to protonation of sulfa drug, the intensity of precursor ion was much higher in positive mode. The optimization of product ions (Q3) and their collision energy were performed using MRM event optimization method. Three product ions for each analyte listed in Table 1 were obtained by MRM optimization method.

Method Validation

The validation parameters for each compound were evaluated individually. The calibration curves generated for the quantification and the confirmation MRMs were linear for all sulfa drugs in a wide range of concentrations, with correlation coefficients (r²) 0.997, 0.998, 0.997, 0.993, 0.996 and 0.998 for STZ, SDZ, SMZ, SMT, SMP and SMX, respectively shown in Table 2. LOD and LOQ values of certified STZ, SDZ, SMZ, SMT, SMP and SMX standard were determined at 0.17, 0.29, 0.12, 0.16, 0.15, 0.27 ng/g and 0.53, 0.87, 0.36, 0.50, 0.95, 0.83 ng/g respectively. The accuracy and precision (co-efficient variations, CV) of the method are listed in Table 3. The accuracy demonstrated as the recovery of each sample shown in Table 3 was calculated from matrix-matched calibration curve. The results gave the recovery of the six tested sulfa drugs at two spiking levels (5 & 10 ng/g) ranging from 82.58 to 109.52 % and CVs that ranged from 1.67% to 15.06% which were within acceptable limit. The repeatability, as represented by intra-day CV percentages, was within 15.06%, which is very satisfactory. Inter-day CV percentages which define reproducibility were providing satisfactory results that all the CV values were lower than 12.15%.

Matrix effect

The matrix enhancement effects for poultry meat samples were 82.41, 76.59, 84.07, 83.11, 76.25 and 82.96% for STZ, SDZ, SMZ, SMT, SMP and SMX, respectively. Again, the matrix enhancement effects for beef samples were 84.51, 85, 84.75, 79.29, 81.36 and 84.56% for STZ, SDZ, SMZ, SMT, SMP and SMX, respectively. So, matrix matched calibration curves were used for quantitative analysis.

Analysis of real samples

The applicability of the described method for routine analysis in food was investigated through the analyses of the selected sulfa drugs in meat samples. Thirty poultry meat and thirty beef samples were collected from different thirty markets and supermarkets in Dhaka city and analyzed (each three replicates) by validated LC-MS/MS method. From Fig. 2 it can be decided that sulfa drugs residue was below detection limit in all selected samples. Though sulfa drugs residue found below the detection limit, upon regular exposure drug residues may accumulate on animal tissue which may lead to drug resistance activity³³. It is reported that sulfa drugs may degrade by enzymes found in animal and microorganisms. Sulfa drugs are polar compounds and excreted by animals through various metabolisms, for example, N-acetylation³⁴. Results from recent research work showed sulfa drugs residue has been detected in manure³⁵. Manure of the waste of food animals from the agricultural field was up-taken by vegetables, and radish is grown in that soil³⁶. Moreover, it has been recommended that the extraction procedure is performed immediately after the homogenization of the sample in order to prevent analyte loss because of the reaction with endogenous enzymes³⁴.

Poultry meat and beef are the major source of meat as well as protein throughout the world. An analytical method based on QuEChERS extraction followed by analysis through LC-MS/MS was validated in this study for the routine analysis of sulfa drugs residue in meat samples. This method is easy, fast, selective, precise, and accurate as well as in comparison to other methods based on HPLC-MS/MS; this method appears to be less expensive than³⁷ which uses affinity chromatography for sample purification³⁸. The developed method was found to be effective and reliable for the analysis of antibiotics residue in meat by LC-MS/MS. This method can be used to improve food safety in Bangladesh. The development of a selective and sensitive method is very essential as it may open the door for an area of contaminant analysis. The validation of the method was performed in poultry meat and beef muscle, but also applicable to egg, feed, milk, liver and kidney samples as well. In this study, sulfa drugs residue in poultry meat and beef samples were found to be present below the detection limit. The study showed that beef and poultry meat available in the market is safe for consumption. But this is a limited study to draw a firm conclusion, a large number of samples need to be analyzed. In a limited period, it was not possible to perform such studies. Moreover, studies of the stability of the analyte in matrix are also important besides real sample analysis.

Acknowledgments

The authors are grateful to the Higher Education Quality Enhancement Project (HEQEP), UGC Dhaka, Bangladesh, and International Science Programme (ISP) Uppsala University, Sweden for financial support.

Declaration of competing interest

The authors declare that they have no competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Author contributions

All authors listed have equal contributions to the work, manuscript preparation, revision, and approved the submitted version for publication.

Data availability

The datasets generated and/or analyzed during the current study cannot be shared publicly but it’s available to the corresponding author. The raw data generated in this study are processed using different equations to meet our required validation parameters. According to our research group policy, raw data sets are not open publicly during the Ph.D. study of a student. This work was carried out for the dissertation/thesis, which is not submitted yet. She is doing more work by using this method with modifications in the different matrices and will include it in her thesis. So, it will be helpful if our raw data are not publicly available.

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Table 1 | MS/MS parameters on the precursor and quantitative product ion(m/z) and collision energy of six sulfa drugs.

Name	Retention time (min)	MRM method (m/z)		Voltage (V)
Name	Retention time (min)	Precursor ion [M-H]⁺	Product ion	Q1 Pre Bias	CE	Q3 Pre Bias
STZ	2.41	256.0	92.10	20.0	18.0	22.0
			108.0	20.0	21.0	25.0
			156.0	20.0	23.0	18.0
SDZ	2.42	251.0	92.00	25.0	30.0	18.0
			108.10	25.0	28.0	20.0
			156.00	25.0	22.0	23.0
SMZ	2.91	265.0	92.10	30.0	25.0	32.0
			108.10	30.0	27.0	29.0
			156.05	30.0	28.0	29.0
SMT	3.59	279.0	92.0	18.0	20.0	23.0
			108.00	18.0	19.0	27.0
			156.0	18.0	17.0	21.0
SMP	3.61	281.0	92.00	19.0	22.0	27.0
			107.90	19.0	25.0	28.0
			156.05	19.0	20.0	25.0
SMX	6.18	311.0	92.0	17.0	26	25.0
			156.0	17.0	23	21.0
			108.10	17.0	19	20.0

Table 2 | MRM data acquisition parameters for the six sulfa drugs using LC-MS/MS

Compound Name	Mass analyzer		Product ion, Q3, m/z (used in analysis)	Linear range (ng/g)	R² (matrix matched)	R² (mobile phase)	LOD (ng/g)	LOQ (ng/g)
Compound Name	Precursor ion,Q1, m/z	Product ion, Q3, m/z	Product ion, Q3, m/z (used in analysis)	Linear range (ng/g)	R² (matrix matched)	R² (mobile phase)	LOD (ng/g)	LOQ (ng/g)
STZ	256.0	92.10 108.00 156.00	92.10	1.25-20	0.997	0.999	0.17	0.53
SDZ	251.0	92.00 108.10 156.00	108.10		0.998	0.999	0.29	0.87
SMZ	265.0	92.00 108.10 156.00	108.10		0.997	0.999	0.12	0.36
SMT	279.0	92.10 108.00 156.00	156.00		0.993	0.999	0.16	0.50
SMP	281.0	92.10 108.00 156.05	108.00		0.996	0.999	0.15	0.95
SMX	311.0	92.10 107.90 156.00	92.10		0.998	0.999	0.27	0.83

Table 3 | Validation parameters of the six sulfa drugs in chicken and beef samples

Name	Sample	Spiking level (ng/g)	Intra-day-1 (n=5)		Intra-day-2 (n=5)		Intra-day-3 (n=5)		Inter Day (n=15)
Name	Sample	Spiking level (ng/g)	Recovery (%)	CV (%)	Recovery (%)	CV (%)	Recovery (%)	CV (%)	Recovery (%)	CV (%)
STZ	Chicken	5	109.43	1.67	104.23	4.15	104.66	3.32	106.11	3.04
	Chicken	10	97.44	2.73	97.69	2.15	98.85	3.39	97.99	2.76
	Beef	5	106.07	2.48	93.00	15.06	101.88	2.96	100.32	6.84
	Beef	10	103.65	2.73	88.43	7.20	82.58	7.71	91.56	5.88
SDZ	Chicken	5	109.52	5.72	105.07	5.96	103.60	6.05	106.06	5.91
	Chicken	10	96.72	3.24	96.96	3.23	104.10	3.01	99.26	3.16
	Beef	5	107.27	3.67	103.60	3.39	105.92	3.31	105.60	3.46
	Beef	10	98.33	6.28	94.44	6.08	87.28	4.48	93.35	5.61
SMZ	Chicken	5	105.25	6.05	103.71	6.14	107.38	5.93	105.45	6.04
	Chicken	10	96.83	3.29	98.03	3.25	98.69	3.22	97.85	3.25
	Beef	5	106.32	5.89	104.98	5.97	109.52	5.72	106.94	5.86
	Beef	10	104.26	3.00	98.59	3.18	90.52	3.46	97.79	3.21
SMT	Chicken	5	107.96	7.33	103.16	7.67	103.02	7.68	104.71	7.56
	Chicken	10	92.81	4.26	96.58	4.09	97.01	4.08	95.46	4.14
	Beef	5	106.32	5.99	106.68	5.97	102.98	6.18	105.32	6.05
	Beef	10	104.35	3.05	98.31	3.24	96.67	3.29	99.77	3.19
SMP	Chicken	5	106.81	11.92	106.29	11.98	101.54	12.54	104.88	12.15
	Chicken	10	98.09	6.49	96.66	6.59	99.01	6.43	97.92	6.50
	Beef	5	104.89	7.54	104.86	7.54	108.50	7.29	106.08	7.46
	Beef	10	104.16	3.80	98.50	4.02	93.01	4.25	98.55	4.02
SMX	Chicken	5	103.88	12.26	105.43	12.08	105.55	12.07	104.95	12.14
	Chicken	10	92.00	6.92	95.50	6.73	98.32	6.47	95.27	6.71
	Beef	5	106.21	11.99	104.27	12.21	105.19	12.11	105.22	12.10
	Beef	10	97.88	6.51	89.54	7.12	97.55	6.53	94.99	6.71

No competing interests reported.

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Validation of a Method for Analysis of Sulfa Drugs in Poultry and Beef Samples using Liquid Chromatography Tandem Mass Spectrometry

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