Beef and Raw and Liquid Chromatography

Toxins (Basel). 2020 Jun; 12(vi): 357.

Multi Mycotoxin Decision in Stale Beef Using Liquid Chromatography Coupled with Triple Quadrupole Mass Spectrometry (LC-MS/MS)

Toluwase Adeseye Dada

^oneSection of Animal Health, Faculty of Natural and Agricultural Sciences, Mafikeng Campus, North West University, Individual Bag X2046, Mmabatho 2735, Mafikeng, South Africa; az.ca.uwn@49351132

²Ekiti State College of Agriculture and Technology, Isan Ekiti 371106, Ekiti State, Nigeria

Theodora Ijeoma Ekwomadu

¹Section of Animal Health, Kinesthesia of Natural and Agricultural Sciences, Mafikeng Campus, North West University, Individual Bag X2046, Mmabatho 2735, Mafikeng, South Africa; az.ca.uwn@49351132

Mulunda Mwanza

¹Department of Animal Health, Faculty of Natural and Agronomical Sciences, Mafikeng Campus, North West University, Private Bag X2046, Mmabatho 2735, Mafikeng, Southward Africa; az.ca.uwn@49351132

Received 2020 April 10; Accepted 2020 May 11.

Abstract

Dried beef meat, a locally processed meat from the cow, is vulnerable to contamination by mycotoxins, due to its exposure to the environmental microbiota during processing, drying, and betoken of sale. In this study, 108 dried beef samples were examined for the occurrence of 17 mycotoxins. Samples were extracted for mycotoxins using solid-liquid stage extraction method, while liquid chromatography coupled with triple quadrupole mass spectrometry (LC-MS/MS) via the dilute and shoot method was used to clarify the mycotoxins. Aflatoxin was institute in 66% of the samples (average value of 23.56 µg/kg). AFB₁ had a mean value of 105.4 µg/kg, AFB2 hateful value of six.92 µg/kg, and AFG_i and AFG₂ had an average mean value of 40.49 µg/kg and 2.60 µg/kg, respectively. The total aflatoxins exceed the EU (iv μg/kg) permissible level in food. The α-Zea average mean value was 113.82 µg/kg for the diverse selling locations. Also, cyclopiazonic acid had an average hateful value of 51.99 µg/kg, while some of the beef samples were contaminated with more than 9 different mycotoxins. The occurrence of these mycotoxins in dried beefiness is an indication of possible exposure of its consumers to the dangers of mycotoxins that are usually associated with severe health bug. This effect shows that in that location are mycotoxin residues in the beef sold in Ekiti Land markets.

Keywords: aflatoxins, mycotoxins, liquid chromatography, mass spectrometry, ochratoxin A, solid-liquid extraction, zearalenone, stale beefiness

ane. Introduction

Mycotoxins contamination in food has remained a major business concern in food condom as they are toxic secondary metabolites produced by several molds and often contaminate food and feed worldwide [one]. Attributable to the likely natural contamination of grains used past the nutrient and livestock manufacture, there is a loftier probability of mycotoxins to enter the food chain [2,3]. Their incidence is influenced by various factors, such as the food product type, climatic weather condition, agricultural practices, storage weather, and periodic variances [4,five]. Mycotoxins have the potential to affect human health by acute and chronic effects, such as the induction of hepatocellular carcinoma (HCC) or unexpected death due to acute toxicity in the case of aflatoxins [6].

Prior research has revealed that products of brute sources, for example meat and meat products, tin can also increase the homo mycotoxins' intake coming either from indirect transfer from farm animals exposed to naturally contaminated grains and feed (carry-over furnishings) or straight contamination with fungi, or naturally contaminated seasoning blends used in meat production [7,viii]. Amid mycotoxins of health importance in relation to their toxicity and incidence, aflatoxin (AFs), ochratoxin A (OTA) fumonisins (FBs), and zearalenone (ZEN) are the most oftentimes encountered contaminants [9]. AFB_one is the most potent liver carcinogen recognized in mammals, classified past the International Agency for Research on Cancer (IARC) as a grouping ane carcinogen, OTA in grouping 2B, and ZEN in grouping 3 carcinogens [10].

The occurrence of mycotoxins in food and feed is potentially hazardous to the health of humans in addition to animals, because of their carcinogenic, toxic, and mutagenic properties [11]. Among farm animals, pigs are known to be predominantly sensitive to mycotoxins, while ruminants such equally sheep and cows are less predisposed, as their rumen fluid is capable of enzymatically degrading these mycotoxins into less toxic metabolites [7]. It has been stated [12] that the allowed modulation furnishings of some mycotoxins increase health impacts of major illnesses disturbing Africa, such as malaria, kwashiorkor, and HIV/AIDS. Information technology is credible that sub-Saharan residents are decumbent to prolonged dietary mycotoxin exposure since they regularly eat afflicted crops, and due to the fact that crops in tropical and subtropical areas are more vulnerable to contamination owing to the suitable climatic conditions [xiii,14].

A number of studies have shown that fungi species belonging to Penicillium and Aspergillus genera are producers of these mycotoxins, and take been isolated from meat products, such equally processed sausages or dry-cured hams [xv,xvi,17]. There are other studies that showed that meaning OTA levels are likely to be found in meat products produced from contaminated raw materials [iii,8], as well as in smoked meat and meat products [18,19]. It is likewise acknowledged that aflatoxins can exist institute in meat as well every bit in meat products, if the animals consume sufficient amounts of AFB₁ occurring in their feed [xv,20,21].

In Nigeria, the consumption of dried meat is increasing especially in the due south-west region and in that location are studies indicating the presence of fungi capable of producing mycotoxins on the dried meat from other states in the south-west region [22,23]. This might exist every bit a result of the preservation method that is unremarkably employed to lower the water activity such as dehydration, and the addition of salt in some cases. All the same, equally the meat surfaces dry out, there is increased possibility of growth of undesirable fungi, several of which can produce potent mycotoxins, due to the favorable weather during the drying, storage, and selling process. The presence of fungi on regular food items in Nigeria is not in incertitude, since the prevalence of mycotoxins in sub-Saharan Africa has been reported [24,25]. However, there is a dearth of information on multi-mycotoxins in stale beefiness in Nigeria while a lot of attention is placed on other food products. In lieu of the above facts, the present report is aimed at investigating the level of AFs, OTA, and ZEN using liquid chromatography coupled with triple quadrupole mass spectrometry (LC-MS/MS) to assess a wider range of potential mycotoxins that contaminates dried meat sold in Ekiti State destined for man consumption.

2. Results and Discussion

2.i. Method Validation

The results obtained from the validation method used in the sample extraction and LC-MS method in the determination of mycotoxins in the dried beef samples is shown in Table 1 and Tabular array 2. Recovery values ranged from 114 to 130% for aflatoxin B_ane, B_two, K_one, and M₂. For sterigmatocystin (STE) and OTA, the recovery ranged from lxx to 73%, while HT-ii had 78–81% recovery values. The matrix furnishings (ME) evaluation was performed using matrix-matched calibration curves; the suppression of the signal (SS) obtained for all the mycotoxins ranged from sixteen to 87%. The obtained limits of detection (LOD) and limits of quantification (LOQ) values for each mycotoxin ranged between 0.xiii µg/kg (OTA)–127 µg/kg (α-ZEA) and 0.4 µg/kg (OTA)–385 µg/kg (α-ZEA), respectively. For the evaluation of the linearity, calibration curves were constructed for all mycotoxins at twelve different levels of concentrations from 0.9 to 2000 µg/kg for all the mycotoxins. The validation results showed strong correlation coefficients (Rⁱⁱ > 0.992). The intra-day accuracy was evaluated by nine determinations at each count level of morning, afternoon, and night on the same day, while inter-day precision was adamant for three days. The range of relative standard deviations was betwixt one.81% and sixteen.4% for the intra-solar day precision, and between 2.12% and fifteen.1% for the inter-twenty-four hour period precision. The recovery results ranged from 70% to 130%, except for ochratoxin B (OTB), T-2, 3-ACDON and tenuazonic copper salt (TCS) (36–41, 58–64, 48–57, 11–52) respectively.

Table ane

Multiple reaction monitoring transitions, mass spectrometer conditions, and retentivity times of the determined mycotoxins.

South/No	Mycotoxin	Ret. Fourth dimension (min)	Precursor (mz)	Products (mz)	Q1 Pre Bias (V)	CE	Q3 Pre Bias (V)
1	3ACDON	6.589	338.9	231	−24	−15	−22
				213	−12	−17	−20
2	HT-two	8.817	425	263	−20	−12	−eighteen
				104.nine	−sixteen	−47	−xix
3	AFG2	7.507	331	245.i	−12	−32	−24
				313	−12	−24	−20
4	AFG1	seven.767	329	243	−12	−28	−23
				311.i	−xvi	−24	−14
5	AFB1	8.25	313	241	−22	−41	−23
				285.1	−22	−24	−29
6	B-ZEA	8.949	323.one	277.2	−16	−sixteen	−xviii
				305.2	−16	−11	−20
vii	a-ZEA	nine.415	323.ane	277.ii	−17	−17	−eighteen
				305.2	−24	−9	−twenty
8	T-2 Toxin	9.674	467.2	245.1	−13	−11	−xvi
				305.ii	−22	−11	−20
nine	ZEA	10.063	319.1	185	−12	−27	−30
				187.i	−15	−21	−19
x	AME	10.125	273	128.1	−10	−49	−21
				115.05	−18	−54	−xix
11	OTB	9.331	370.i	205	−xiii	−22	−21
				324.1	−13	−14	−22
12	OTA	10.132	403.8	239	−fifteen	−27	−24
				221	−12	−38	−21
xiii	AFB2	8.007	315	259.1	−22	−31	−25
				287	−23	−26	−30
14	TCS	5.02	198.15	125.05	−10	−18	−21
				-	-	-	-
15	Citrinin	5.00	251	205.1	−thirteen	−26	−21
				233	−17	−17	−24
16	STECY	5.01	324.xc	310	−22	−24	−thirty
				281.one	−22	−twoscore	−27
17	CPA	five.01	337.1	196.1	−x	−23	−18
				182.1	−10	−xx	−xx

Table ii

Analytical parameters for liquid chromatography coupled with triple quadrupole mass spectrometry (LC-MS/MS) method validation: matrix event, linearity, limits of detection (LOD), limits of quantitation (LOQ), recoveries at iii spiked concentration levels, intra-day, and inter-mean solar day precision for the studied mycotoxins.

Mycotoxin	Matrix Result (%)	Linearity r^two	LOD µg/kg	LOQ µg/kg	Recovery % Spiked Level (µg/kg)			Intra-twenty-four hours(RSD) % Spiked Level (µg/kg)			Inter-twenty-four hours (RSD) % Spiked Level (µg/kg)
					25	50	100	25	50	100	one	3	5
AFB₁	73	0.998	two	6.8	114	121	124	10.9	6.5	4.8	4.22	3.71	three.75
AFB₂	75	0.999	6	17	121	125	127	4.eight	2.7	3.xv	three.42	iv.35	3.59
AFG_ane	77	0.997	6	16	130	129	130	6.68	6.73	three.07	3.63	4.48	3.69
AFG_ii	83	0.994	44	134	114	120	126	8.2	9.03	iii.63	3.57	2.12	iii.47
STER	47	0.999	viii	23	73	71	71	iii.71	half dozen.89	xiii.1	13.2	xv.one	14.7
OTA	60	0.997	0.13	0.4	71	70	seventy	12.4	5.01	three.29	5.xviii	5.38	6.81
OTB	48	0.999	0.41	i.2	36	36	41	5.43	5.47	seven.33	9.51	5.81	vi.46
ZEA	73	0.999	123	374	108	113	114	5.47	v.56	4.7	3.12	5.38	3.1
T-2	52	0.992	84	254	59	64	58	4.64	half dozen.31	iii.85	6.xix	6.09	6.14
HT-two	35	0.999	iii	10	81	79	78	sixteen.iv	vii.62	4.59	four.86	4.62	iv.58
3-ACDON	39	0.997	25	75	48	52	57	two.33	10.one	6.92	6.94	6.18	6.28
AME	66	0.994	55	166	112	114	111	4.41	3.85	ii.12	ii.34	2.45	two.71
α-ZEA	72	0.998	127	385	117	116	114	iii.49	5.54	2.57	3.29	3.55	3.3
β-ZEA	70	0.998	19	57	102	99	99	ii.75	four.42	one.81	two.49	3.1	3.37
Citrinin	87	0.998	18	54	101	107	107	2.17	2.half-dozen	three.09	four.59	3.33	4.71
CPA	75	0.996	vii	xx	81	71	71	5.23	4.61	three.15	half dozen.91	6.12	half dozen.11
TCS	43	0.998	8	25	53	26	11	viii.39	five.23	5.65	6.38	6.27	half dozen.21

2.2. Occurrence of Mycotoxins in Dried Beef

The analyzed samples in the current study were contaminated with at least four different types of mycotoxins, while about 60% of samples showed co-occurrence of iv to 10 dissimilar mycotoxins. The occurrence of mycotoxins in the samples are shown in Table 3. The CPA was the near prevalent mycotoxin which was detected in 94% of the samples with an average mean of 51.99 ppb, followed by AFB₁ with 50%, ranging from three.91 to 295.41 with an average hateful of 95.25 ppb, AFB_two has (38% with a range of 0.65–33.13 and an boilerplate mean value of 6.93 ppb, AFG_one and α-ZEA recording (33%) with a different average mean value of forty.49 and 113.82 ppb, STE (22%) mean value was v.09 ppb, OTA (xvi%) has mean value of 0.54 ppb, TCS (xiv%) has the mean value of thirteen.58 ppb, OTB (13%) has an boilerplate hateful value of 0.04 ppb, and AFG_two (eight%) recorded an average mean value of ii.61 ppb. Withal, HT-ii, β ZEA, CIT, T2, alternariol monomethyl ether (AME), and 3-ACDON were not detected in any of the samples.

Table 3

Occurrence (%) of mycotoxins in analyzed samples from different locations (north = 108).

Location	HT 2	CPA	TCS	AFB₁	AFB₂	AFG₁	AFG_two	ZEA	α ZEA	β ZEA	CIT	T2	OTA	OTB	STER	AME
Ikole	0(fifteen)	fourteen(xv)	four(15)	8(xv)	ix(xv)	vii(15)	iv(15)	0(15)	6(xv)	0(15)	0(15)	0(15)	ane(15)	iii(15)	half dozen(15)	0(15)
Ilawe	0(six)	5(half dozen)	1(6)	i(6)	0(6)	0(six)	0(half dozen)	0(half dozen)	2(vi)	0(6)	0(6)	0(6)	0(6)	0(half-dozen)	0(half-dozen)	0(6)
Aramoko	0(12)	12(12)	ane(12)	half dozen(12)	2(12)	7(12)	0(12)	0(12)	5(12)	0(12)	0(12)	0(12)	1(12)	1(12)	ane(12)	0(12)
Ijero	0(ten)	x(10)	0(x)	7(10)	v(ten)	4(x)	0(ten)	0(10)	2(10)	0(10)	0(10)	0(10)	5(10)	one(10)	4(ten)	0(10)
Oye	0(half-dozen)	six(6)	3(6)	4(6)	2(vi)	0(6)	1(6)	0(half dozen)	2(6)	0(half dozen)	0(6)	0(6)	3(6)	1(6)	2(six)	0(six)
Otun	0(fifteen)	xiii(fifteen)	0(xv)	four(15)	2(fifteen)	four(fifteen)	1(xv)	0(15)	5(15)	0(xv)	0(fifteen)	0(15)	0(15)	ane(xv)	iii(15)	0(15)
Ado	0(xx)	twenty(20)	3(20)	12(20)	xi(xx)	6(twenty)	0(20)	0(20)	v(20)	0(20)	0(20)	0(20)	6(20)	iv(20)	5(20)	0(20)
Omuo	0(14)	thirteen(fourteen)	2(14)	7(14)	5(14)	4(fourteen)	i(fourteen)	0(14)	five(14)	0(xiv)	0(fourteen)	0(14)	one(14)	2(14)	2(xiv)	0(14)
Igede	0(6)	6(half-dozen)	1(6)	iv(6)	3(vi)	3(6)	2(half dozen)	0(six)	2(half dozen)	0(6)	0(half dozen)	0(6)	0(six)	1(half dozen)	1(6)	0(half-dozen)
Ise/Emure	0(4)	3(four)	0(4)	1(4)	two(4)	one(4)	0(iv)	0(4)	2(4)	0(4)	0(4)	0(4)	0(4)	0(4)	0(4)	0(4)
Total %	0%	102 94%	15 14%	54 50%	41 38%	36 33%	9 8%	0%	36 33%	0%	0%	0%	17 16%	14 13%	24 22%	0%

2.3. Co-Occurence of Mycotoxins in Meats from the Different Sampling Locations

The occurrence based on mean concentration of each mycotoxin are shown co-ordinate to the different locations in Effigy 1. Contamination levels of AFB₁ were higher and were similar in Ikole and Igede marketplace, followed past α-ZEA, and then AFG_one in Igede market. In Ilawe, Aramoko, Otun, Omuo, and Ise/Emure, α-ZEA was the ascendant mycotoxin. Ilawe and Ise/Emure samples had the lowest mycotoxin co-contamination of four and 6 out of the 18 samples analyzed for mycotoxins, respectively, followed by Ijero and Otun, both with 8 different types of mycotoxins in that order. Ikole and Omuo had 10 different types of mycotoxins that are present in at least one of the samples, this make them the highest locations of simultaneous mycotoxin contaminations, followed by Aramoko, Oye, Ado, and Igede, with each recording 9 different mycotoxins separately. This trend of contamination could exist equally a issue of unlike fungi peculiarity to the different locations where the cattle were fed and reared, as the meat samples were sourced from virtually 3 different states from the Northern part of Nigeria. This could also be influenced by storage, air pollution, and human contact with the product, every bit well as displaying method. Foods are frequently contaminated with several fungi, and one time the temperature and relative humidity is at the peak later contagion, there might be likely mycotoxin production [5]. It has also been established that relatively low water activity (aw < 0.9) with low pH values that is less than half dozen.0 are principally favorable for fungi growth [26]. The dried meat samples analyzed had low water activity and pH boilerplate value of 5.0. This is congruent with favorable h2o activity and pH values known to favor fungi and mycotoxin production, hence the probable level of fungi contamination and mycotoxin level discovered in the stale meats.

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Concentration of mycotoxins in dried beef from different locations/markets.

Previous studies showed that mycotoxin contamination in both raw and processed pork meat products is common [15,27]. Although mycotoxins naturally occur in meat primarily as a effect of an indirect transfer from naturally contaminated feed [28], production, transportation, storage, and displaying conditions tin also influence the occurrence of mycotoxins on dried meat sold in open markets where they are not protected from their environs.

ii.four. Trichothecenes Level in Dried Beef

Recovery for three-ACDON and T-two was low (48–57% and 58–64%) respectively, with the exception of HT-2 which recorded a recovery charge per unit of 78–81%. The other trichothecenes were detected at depression concentrations and depression occurrences. It was difficult to compare the obtained data with other studies due to the limited literature available.

2.5. Ochratoxin A Level in Dried Beef

The event showed that OTA was detected in 14% (n = 108) of the meat samples at concentrations ranging from 0.02 to 3.96 µg/kg, with an average mean of 0.5 µg/kg. OTA contamination levels observed herein are comparable to the one reported past [29], where mean value for OTA in French delicatessen meats was 0.25 µg/kg, but lower than the OTA mean value of v.23 µg/kg reported by [xxx] in beef luncheon and four.55 µg/kg in beef burger samples respectively. This study showed that ochratoxin A levels in dried beef from Ekiti Land were lower than the maximum permissible level (1 μg/kg) stipulated for pork products in some European union (Eu) countries [three].

ii.half-dozen. Aflatoxin Total Level in Dried Beefiness

The total aflatoxin content detected in this written report, every bit show in Tabular array four and Figure two, showed that AFB_i values ranged between three.91 and 295.41 µg/kg with a mean value of 105.4 µg/kg, and AFB₂ ranged from 0.65 to 33.13 µg/kg with mean value of six.92 µg/kg. AFG₁ and AFG_ii ranged from 2.24 to 257.35 µg/kg and 0.38–19.08 µg/kg with mean value of 40.49 µg/kg and 2.60 µg/kg, respectively. The range and hateful value recorded in the present written report is higher than the range of 1.10–8.32 µg/kg and 0.15–6.36 µg/kg reported previously [twenty]. AFB₁ is the predominant contaminant amidst the AFs, contaminating virtually fifty% of the beefiness samples analyzed, which is far college than the 10% reported in chicken meat from Croatia [9]. Information technology exceeded the maximum permissible level (MPLs) adopted by over 75 countries around the earth for AFB₁ and full aflatoxins of 5 and ten µg/kg, respectively [3], and not more 2 and iv µg/kg stipulated by the European Union for AFB_ane and total aflatoxins [20].

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Total mean mycotoxin concentration in analyzed stale beef samples in Ekiti State.

Table 4

Concentration (µg/kg) of mycotoxins in analyzed dried meat samples from different locations in Ekiti State (n = 108).

Location	Ikole	Ilawe	Aramoko	Ijero	Oye	Otun	Ado	Omuo	Igede	Ise/Emure
HT 2	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND
CPA	118.87 ± 52.05 ^b	6.42 ± 4.74 ⁱ	13.36 ± five.11 ^g	25.81 ± x.54 ^f	163.93 ± 94.75 ^a	10.36 ± eight.51 ^h	92.62 ± 44.61 ^c	56.43 ± 38.35 ^d	30.03 ± 14.90 ^e	2.xi ± 1.11 ^j
TCS	35.88 ± 28.74	0.30 ± 0.30	55.83 ± 55.83	0	ix.27 ± 4.47	0	eighteen.79 ± eleven.19	15.6 ± fourteen.93	0.16 ± 0.sixteen	0
AFB₁	282.75 ± 136.66	5.85 ± 5.85	15.half-dozen ± 9.62	66.57 ± 45.71	14.ane ± eight.87	59.81 ± 58.53	137.87 ± 60.05	70.69 ± 51.14	295.41 ± 272.73	3.91 ± 3.91
AFB_ii	15.69 ± 7.32 ^ab	0	0.65 ± 0.47 ^b	one.72 ± 0.67 ^b	ane.69 ± i.24 ^b	2.5 ± 2.31 ^b	7.42 ± 2.88 ^b	iii.half-dozen ± 2.51 ^b	33.13 ± 31.02 ^a	2.85 ± 2.48 ^b
AFG_one	88.66 ± 39.09 ^b	0	four.94 ± 1.91 ^c	13.95 ± ix.96 ^b	0	6.82 ± v.78 ^c	15.45 ± 8.21 ^b	15.56 ± viii.81 ^b	257.35 ± 208.95 ^a	2.24 ± ii.24 ^d
AFG₂	5.53 ± 2.55 ^b	0	0	0	0.64 ± 0.64 ^b	0.45 ± 0.45 ^b	0	0.38 ± 0.38 ^b	xix.08 ± 14.87 ^a	0
ZEA	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND
α Zea	137.2 ± 49.00	148.89 ± 124.93	167.34 ± 70.53	47.6 ± 39.fourteen	81.64 ± 51.73	100.83 ± 44.62	103.35 ± 44.46	136.96 ± 52.71	lxxx.33 ± l.97	134.11 ± 77.59
β Zea	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND
CIT	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND
T2	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND
OTA	0.02 ± 0.02 ^b	0	0.03 ± 0.03 ^d	0.28 ± 0.10 ^c	3.96 ± 3.sixty ^a	0	1.12 ± 0.64 ^b	0.02 ± 0.02 ^d	0	0
OTB	0.03 ± 0.02	0	0.01 ± 0.05	0.03 ± 0.03	0.03 ± 0.03	0.01 ± 0.00	0.22 ± 0.xix	0.01 ± 0.00	0.01 ± 0.01	0
STER	25.37 ± 13.77	0	2.64 ± 2.64	3.56 ± 1.78	3.06 ± two.38	1.08 ± 0.59	4.36 ± 3.42	4.98 ± four.62	5.89 ± 5.89	0
AME	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND
iii-AC DON	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND

2.vii. Other Mycotoxins in the Dried Beef

Other important mycotoxins detected in the meat samples include cyclopiazonic acrid, sterigmatocystin, and α-ZEA; interestingly, the concentrations of α-ZEA were relatively higher and ranged from 47.sixty µg/kg to 167.34 µg/kg with an boilerplate mean value of 113.82 µg/kg in the beef samples across the various selling locations. The actual data and concentration ranges are displayed in Tabular array 4 and Figure 2. Also, high concentrations of cyclopiazonic acid (from ii.11µg/kg to 163.nine μg/kg) with an boilerplate mean value of 51.99 µg/kg were similarly detected in the beef samples.

ii.8. Potential Health Risks for Consumption of Contaminated Dried Beefiness

Almost all the analyzed beef samples had meaning amounts of different mycotoxins, and well-nigh people in these areas consume the meat products every bit a effeminateness; as such, consumer's exposure to unlike types of mycotoxins is possible. The continuous exposure of consumers to the contaminated dried meat with cyclopiazonic acid (CPA) could result in immunotoxic and hepatotoxic furnishings that target the muscle, hepatic tissue, and spleen organ of humans [31]. In the instance of alpha zearalenone, which is known to be carcinogenic, it causes hormonal imbalance and have effects on reproductive organs [32,33]. The consumption of aflatoxin is known to cause hepatotoxic effects and suppress the immune system, with liver being its target organ [34]. In the case of ochratoxin A, it is carcinogenic, genotoxic, immune-suppressive, nephritic, and causes upper urinary tract diseases, with its target organ being kidney and liver [35]. Sterigmatocystin is genotoxic, cytotoxic, immunotoxic, and carcinogenic, and mainly attacks the liver, immune organization, and kidney [36,37]. Tenuazonic acid (copper table salt) has been described to exert antiviral, antitumor, clarified, cytotoxic, phytotoxic material, and likewise to be highly toxic in living organisms [38].

3. Conclusions

In summary, a comprehensive screening for regulated besides as other mycotoxins was conducted in dried beefiness from Ekiti State for the first time using the modern screening method. Also the efficiency of the LC-MS/MS and extraction solvent employed showed that it was constructive for the determination of (CPA, AFB_1, AFB_two, AFG_ane, AFG_two, α ZEA, STER, OTA, TCS, and OTB) in stale beef. The assay revealed that dried beef samples were contaminated with different mycotoxins across the various locations in Ekiti State. Likewise, the analyzed samples in the electric current study were contaminated with at least four different mycotoxins, while virtually 60% of samples showed co-occurrence of iv to x unlike mycotoxins out of 17 determined mycotoxins. Dried beef samples from Igede and Ikole location had significantly college mean levels of AFB₁, followed by AFG_1. The occurrence or presence of theses mycotoxins and their potential threat to consumer condom should be of bully business organization. The obtained data offers additional material for discussion virtually the incidence of mycotoxins in dried beef and their impact on the food safety. Although farther research and expansive exposure studies might be necessary, the findings take provided a new outlook for food safety policy makers for the production, handling, storage, and display method for selling dried beef products across the country. It is recommended that farther studies be done to know the extent of mycotoxin conduct-over into edible tissues of cow when the animals are fed with contaminated feed.

four. Materials and Methods

4.1. Reagents, Chemicals, and Extraction Kits

Milli-Q quality water (Millipore, Yard.A.S. 67120 Molsheim, France) was used during the whole assay. Formic acrid, acetonitrile, and methanol were of MS grade, and purchased from Merck (Darmstadt, Germany). The Elisa kits used in the present study were purchased from Sigma-Aldrich (Steinem, Federal republic of germany), while distilled water was used, and all other chemicals used were of analytical course.

4.ii. Standards for LC-MS

The standards of aflatoxin B₁ (AFB_ane), aflatoxin B_ii (AFB₂), aflatoxin Chiliad_one (AFG₁), aflatoxin G2 (AFG₂), ochratoxin A (OTA), ochratoxin B (OTB), sterigmatocystin (STE), a-zearalenol (a-ZEA), ꞵ ZEA zearalenone (ZEN), 3-acetyldeoxynivalenol (three-ADON), alternariol monomethyl ether (AME), Kojic Acid (KA), Citrinin, Sterigmatocystin (STER), Cyclopiazonic acid (CPA), Tenuazonic copper common salt (TCS), T-2, and HT-2 toxins were supplied from the National Metrology Establish of Due south Africa (NMISA). The mycotoxins stock solutions were prepared co-ordinate to a previous study [25].

4.3. Sampling

1 hundred and 8 dried beef meat were purchased from selected ten major open markets from X Local Regime Areas of Ekiti State, Nigeria. The markets include Oye, Ilawe, Ise/Emure, Otun, Omuo, Igede, Ikole, Aramoko, and Ado. Sampling locations comprised two cities each from 5 geographical zones North, Southward, Due east, West, and Cardinal. Each sample was collected from random points of trader's trays as 12–14 sub-samples (fifty–100 g) and mixed together to class the majority sample (200–400 g) and pulverized. A fifty-gram representative sample was obtained from each majority and stored at −twenty °C until analyzed.

4.iv. Extraction Process for LC-MS

The representative samples of particle size betwixt 0.five and ane mm were weighed into a 50 mL polypropylene tube (Sarstedt, Nümbrecht, Germany) and enclosed with extraction solvent made upwards of acetonitrile/water/acetic acrid (79:20:i, v/v/5) in 20 mL solvent/ 5g sample. For spiking and recovery experiments, 5 g of samples were used for extraction. Samples were extracted for 90 min at 180 rpm on a Labcon FS16 rotary shaker (Labcon, South Africa), and centrifuged for 15 min at 3500 rpm. The supernatant was carefully transferred into some other centrifuge tube and ten mL of n-hexane was added to breakdown the fatty sample [39]. The mixture was vortexed for 2 min and allowed to settle for twenty min at room temperature before the northward-hexane stage was removed, and diluted with an equal volume of dilution solvent (acetonitrile/water/ acetic acid 79:twenty: 1, v/v/v), filtered through a 0. 22 µm syringe nylon filter (Membrane Solutions, Tokyo) into one.5 mL HPLC vial bottles for injection into the LC-MS/MS organisation [twoscore].

4.5. Instrumentation for LC-MS

A Shimadzu UHPLC musical instrument LC-MS/MS 8030 equipment (Shimadzu Corporation, Tokyo, Nihon), with an ultrafast scan speed of 15,000 u/sec, and a polarity switching of fifteen msec was used for the identification and quantification of the analytes. The chromatograph was a LC-30AD Nexera which was connected to a SIL-thirty AC Nexera machine sampler and a CTO-20 Air-conditioning Prominence Column Oven. The oven was equipped with a Raptor^TM ARC-18 column from Restek (2.7 µm, two.one mm × 100 mm) (Restek Corporation, PA, USA), and maintained at a constant temperature of xl °C. The mobile phases used consisted of (A) 0.1% formic acid in deionized water and (B) 0.1% formic acrid in acetonitrile: methanol (50:50 5/v), and was delivered at a constant flow rate of 0. ii mL/min with a sample injection volume set at 5 µL. The elution gradient program had a full run time of 20 min, and started with x% B for 0.01 min and increased steadily to 95% B in x min, at which point it was kept constant for 3.5 min, and then the initial condition (10% B) was re-established for 1 min, and the column allowed to re-equilibrate for 5 min for the next run.

Following chromatographic separation, the analytes were committed to a Shimadzu triple quad mass spectrometry detector model 8030 (Shimadzu Corporation, Kyoto, Japan) for detection and quantitation of analyte. The ionization source was an electron spray ionization (ESI) operated in positive mode at an issue time of 0.206 sec. Data was acquired by a multiple reaction monitoring (MRM) method at optimized MS conditions for the analyte (Tabular array 1). The interface nebulizing gas flow rate was iii L/min, DL temperature was 250 °C, heat block temperature was 400 °C, and drying gas flow rate was 15 L/min.

4.6. Validation of the Method for LC-MS

The performance characteristics of the method such as matrix consequence, linearity, trueness, and intra-twenty-four hours and inter-twenty-four hour period precision, limits of detection (LOD) and quantification (LOQ) and selectivity were evaluated.

Matrix effects were estimated using a 12 point matrix-matched calibration curves and a corking standard solution calibration curves inside the linear range of 0.ix, to 2000 µg/kg. Signal suppression/enhancement (SSE) values, which were calculated past comparing the slopes of matrix-matched calibrations with those of pure standard solution calibrations, were used for the evaluation of the matrix effects [41].

Calibration curves were generated by plotting the expanse under peak against the concentration of individual mycotoxin in the meat matrices. The method sensitivity was calculated by determining the limit of detection (LOD), which is given every bit LOD = 3.3 $\times \frac{Conc}{Southward / N}$ , and limit of quantification (LOQ) given equally LOQ = 10 $\times \frac{Conc .}{S / N}$ . The LOD and LOQ values of each mycotoxin were determined past mixing the stock standard solutions with the blank matrices.

The recoveries were adamant at three different levels using blank samples with nine replicates per concentration level, the spiking was done at low (25 µg/kg), intermediate (50 µg/kg), and loftier (100 µg/kg) levels of the mycotoxin concentrations. Preparation of samples was carried out in triplicate, as described above. Extraction recovery for each analyte was evaluated by matching the mean peak areas of the samples previously spiked before extraction with that of the samples spiked after extraction.

Precision was evaluated for intra-twenty-four hours and inter-twenty-four hour period in not-contaminated samples using standard improver. The samples were spiked with iii different concentration levels of each standard (25, 50, and 100 µg/kg) and prepared in triplicate for computing the intra-day precision, while two samples spiked with 50 and 100 µg/kg of each standard were analyzed daily in triplicate for five sequent days for inter-day precision. The adding for recovery and precision were based on matrix-matched calibrations. The mycotoxin concentrations (µg/kg) were calculated using calibration curves with mathematical interpolation and multiplication by the sample'south dilution gene.

4.7. Statistical Analysis

The concentrations were calculated based on the average recovery values acquired for each analyte. Statistical analysis was done using IBM SPSS version 25 Software with the statistical significance level at (p < 0.05).

Key Contribution

The almost of import finding of this study was that mycotoxin is been reported for the kickoff time on dried beef sold in Ekiti State at different locations; another important finding is the utilise of the LCMS method for the simultaneous determination of mycotoxins in dried beef. However, significant differences in the contamination patterns were observed between the sampling locations.

Author Contributions

Conceptualization, T.A.D. and M.Yard.; Methodology, T.A.D. and Thou.Thou.; Formal Analysis and Investigation, T.A.D., M.Chiliad., and T.I.Due east.; Writing—Original Typhoon Preparation, T.A.D.; Writing—Review and Editing, T.A.D. and Chiliad.G. All authors accept read and agreed to the published version of the manuscript.

Funding

This research was supported past the North-West University Graduate Research Bursary 2016-2018 and National Research Plant.

Conflicts of Involvement

The authors declare no disharmonize of interest.

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