A novel role of follicle-stimulating hormone (FSH) in various regeneration-related functions of endometrial stem cells.

Follicle-stimulating hormone (FSH) promotes the production and secretion of estrogen, which in turn stimulates the growth and maturation of ovarian follicles. Therefore, consecutive FSH treatment to induce ovarian hyperstimulation (superovulation) is still considered the most cost-effective option for the majority of assisted reproductive technologies (ARTs). However, a relatively high cancellation rate and subsequent low pregnancy outcomes (approximately 15%) are the most challenging aspects of this FSH-based ART. Currently, the main cause for this low implantation rate of FSH-based ART has not yet been revealed. Therefore, we hypothesized that these high cancellation rates with FSH-based superovulation protocols might be associated with the harmful effects of consecutive FSH treatment. Importantly, several recent studies have revealed that tissue-resident stem cell deficiency can significantly reduce cyclic endometrial regeneration and subsequently decrease the pregnancy outcome. In this context, we investigated whether FSH treatment could directly inhibit endometrial stem cell functions and consequently suppress endometrial regeneration. Consistent with our hypothesis, our results revealed for the first time that FSH could inhibit various regeneration-associated functions of endometrial stem cells, such as self-renewal, migration, and multilineage differentiation capacities, via the PI3K/Akt and ERK1/2 signaling pathways both in vitro and in vivo.

Effects of smoking on the tissue regeneration-associated functions of human endometrial stem cells via a novel target gene SERPINB2.

BACKGROUND: Smokers directly inhale mainstream cigarette smoke, which contains numerous known and potential toxic substances, and thus, smoking is expected to have broad harmful effects that cause tissue injury and dysfunction. Interestingly, many studies have suggested that the recent decline in female fertility and increased rate of spontaneous abortion could be associated with increased smoking rates. Indeed, women that smoked for 10 years or more were reported to have a ~ 20% higher infertility rate than women that had never smoked. However, the reasons for the underlying harmful aspects of smoking on female fertility remain a matter of debate. Importantly, a previous study revealed that resident endometrial stem cell deficiency significantly limits the cyclic regeneration potential of endometrium, which, in turn, decreases successful pregnancy outcomes. In this context, we postulated that exposure to mainstream cigarette smoke extracts might decrease female fertility by inhibiting the functions of resident endometrial stem cells. METHODS: We investigated whether cigarette mainstream smoke exposure directly inhibits various tissue regeneration-associated functions of endometrial stem cells, such as self-renewal, migration, pluripotency, and differentiation capacity in vitro. Next, we determined whether SERPINB2 mediates cigarette smoke-induced suppressive effects on various tissue regeneration-associated functions by depleting SERPINB2 expression with specific shRNA targeting SERPINB2. Mice were injected intraperitoneally with low (0.5 mg/kg) or high (1 mg/kg) doses of cigarette smoke extract (10 times for two weeks), and endometrial stem cells were then isolated from mice uterine tissues. RESULTS: We found that exposure to cigarette smoke extracts remarkably suppressed various tissue regeneration-associated functions of endometrial stem cells, such as self-renewal, migration, multilineage differentiation ability, and pluripotency in vitro and in vivo by activating the SERPINB2 gene. Indeed, cigarette smoke-induced inhibitory effects on various endometrial stem cell functions were significantly abolished by SERPINB2 knockdown. CONCLUSIONS: These findings provide valuable information on the harmful effects of cigarette smoking on resident endometrial stem cells and hopefully will facilitate the developments of promising therapeutic strategies for subfertile or infertile women that smoke cigarettes.

Novel roles of luteinizing hormone (LH) in tissue regeneration-associated functions in endometrial stem cells.

Luteinizing hormone (LH) stimulates the synthesis and secretion of the key steroid hormone estrogen, which subsequently promotes ovarian follicular growth and development. Therefore, the administration of exogenous LH to achieve superovulation (multiple ovulations) and an LH surge is commonly used as the most effective therapeutic option in a majority of in vitro fertilization (IVF) clinics. However, a relatively low pregnancy rate (between 20% and 35%) is one of the most challenging aspects of LH-based infertility treatment. Furthermore, the major cause of this low pregnancy rate in LH-based infertility treatment remains unidentified. Recent studies have shown that endometrial stem cell loss or deficiency can significantly decrease tissue regeneration ability during the menstrual cycle and reduce endometrial receptivity. In this context, we postulated that the low pregnancy rates following LH-based ovarian hyperactivation may be the result of the adverse effects of consecutive exogenous LH administration on endometrial stem cells. To the best of our knowledge, this study revealed for the first time that in addition to its previously reported roles in stimulating ovarian functions through the pituitary-gonadal axis, LH brings about the extragonadal suppression of various tissue regeneration-associated functions in endometrial stem cells, such as self-renewal, migration ability, multilineage differentiation potential, and pluripotency/stemness, by inhibiting pro-survival Akt and ERK1/2 signaling pathways in vitro and in vivo, and as a consequence, it decreases the endometrial receptivity.

The impact of fine particulate matter (PM) on various beneficial functions of human endometrial stem cells through its key regulator SERPINB2.

Fine particulate matter (PM) has a small diameter but a large surface area; thus, it may have broad toxic effects that subsequently damage many tissues of the human body. Interestingly, many studies have suggested that the recent decline in female fertility could be associated with increased PM exposure. However, the precise mechanisms underlying the negative effects of PM exposure on female fertility are still a matter of debate. A previous study demonstrated that resident stem cell deficiency limits the cyclic regenerative capacity of the endometrium and subsequently increases the pregnancy failure rate. Therefore, we hypothesized that PM exposure induces endometrial tissue damage and subsequently reduces the pregnancy rate by inhibiting various beneficial functions of local endometrial stem cells. Consistent with our hypothesis, we showed for the first time that PM exposure significantly inhibits various beneficial functions of endometrial stem cells, such as their self-renewal, transdifferentiation, and migratory capacities, in vitro and in vivo through the PM target gene SERPINB2, which has recently been shown to be involved in multiple stem cell functions. In addition, the PM-induced inhibitory effects on the beneficial functions of endometrial stem cells were significantly diminished by SERPINB2 depletion. Our findings may facilitate the development of promising therapeutic strategies for improving reproductive outcomes in infertile women.

Noncanonical functions of glucocorticoids: A novel role for glucocorticoids in performing multiple beneficial functions in endometrial stem cells.

Chronic stress has a negative impact on many fertility-related functions; thus, the recent decline in female fertility seems to be at least partially associated with increased stress. The secretion of glucocorticoids is a typical endocrine response to chronic stress and indirectly reduces uterine receptivity through the hypothalamus-pituitary-gonadal (HPG) axis. However, in addition to its well-known canonical role, the direct effects of chronic stress-induced glucocorticoids on various uterine functions and their underlying molecular mechanisms are complex and have not yet been revealed. Recent studies have found that resident stem cell deficiency is responsible for the limited regenerative potential of the endometrium (the innermost lining of the uterine cavity) during each menstrual cycle, which subsequently increases infertility rates. In this context, we hypothesized that stress-induced glucocorticoids directly damage endometrial stem cells and consequently negatively affect endometrial reconstruction, which is important for uterine receptivity. In addition to its well-known canonical roles, we identified for the first time that cortisol, the most abundant and potent glucocorticoid in humans, directly suppresses the multiple beneficial functions (self-renewal, transdifferentiation, and migratory potential) of human endometrial stem cells through its functional receptor, glucocorticoid receptor (GR). Glucocorticoids inhibit well-known survival signals, such as the PI3K/Akt and FAK/ERK1/2 pathways. More importantly, we also found that immobilization of stress-induced glucocorticoids suppresses the various beneficial functions of tissue resident stem cells in vivo. To the best of our knowledge, this is the first study to investigate the direct effects of glucocorticoids on the regenerative capacity of endometrial stem cells, and the findings will facilitate the development of more promising therapeutic approaches to increase female fertility.

Development of an analytical method for multi-residue quantification of 18 anthelmintics in various animal-based food products using liquid chromatography-tandem mass spectrometry.

In this study, we developed a simple screening procedure for the determination of 18 anthelmintics (including benzimidazoles, macrocyclic lactones, salicylanilides, substituted phenols, tetrahydropyrimidines, and imidazothiazoles) in five animal-derived food matrices (chicken muscle, pork, beef, milk, and egg) using liquid chromatography-tandem mass spectrometry. Analytes were extracted using acetonitrile/1% acetic acid (milk and egg) and acetonitrile/1% acetic acid with 0.5 mL of distilled water (chicken muscle, pork, and beef), and purified using saturated n-hexane/acetonitrile. A reversed-phase analytical column and a mobile phase consisting of (A) 10 mM ammonium formate in distilled water and (B) methanol were used to achieve optimal chromatographic separation. Matrix-matched standard calibration curves (R 2 ≥0.9752) were obtained for concentration equivalent to ×1/2, ×1, ×2, ×3, ×4, and ×5 fold the maximum residue limit (MRL) stipulated by the Korean Ministry of Food and Drug Safety. Recoveries of 61.2-118.4%, with relative standard deviations (RSDs) of ≤19.9% (intraday and interday), were obtained for each sample at three spiking concentrations (×1/2, ×1, and ×2 the MRL values). Limits of detection, limits of quantification, and matrix effects were 0.02-5.5 µg/kg, 0.06-10 µg/kg, and -98.8 to 13.9% (at 20 µg/kg), respectively. In five samples of each food matrix (chicken muscle, pork, beef, milk, and egg) purchased from large retailers in Seoul that were tested, none of the target analytes were detected. It has therefore been shown that this protocol is adaptable, accurate, and precise for the quantification of anthelmintic residues in foods of animal origin.

Development of an analytical method for multi-residue quantification of 18 anthelmintics in various animal-based food products using liquid chromatography-tandem mass spectrometry / 药物分析学报

In this study,we developed a simple screening procedure for the determination of 18 anthelmintics(including benzimidazoles,macrocyclic lactones,salicylanilides,substituted phenols,tetrahydropyr-imidines,and imidazothiazoles)in five animal-derived food matrices(chicken muscle,pork,beef,milk,and egg)using liquid chromatography-tandem mass spectrometry.Analytes were extracted using acetonitrile/1％acetic acid(milk and egg)and acetonitrile/1％acetic acid with 0.5 mL of distilled water(chicken muscle,pork,and beef),and purified using saturated n-hexane/acetonitrile.A reversed-phase analytical column and a mobile phase consisting of(A)10 mM ammonium formate in distilled water and(B)methanol were used to achieve optimal chromatographic separation.Matrix-matched standard calibration curves(R2≥0.9752)were obtained for concentration equivalent to ×1/2,×1,×2,×3,×4,and ×5 fold the maximum residue limit(MRL)stipulated by the Korean Ministry of Food and Drug Safety.Recoveries of 61.2-118.4％,with relative standard deviations(RSDs)of ≤19.9％(intraday and interday),were obtained for each sample at three spiking concentrations(×1/2,×1,and ×2 the MRL values).Limits of detection,limits of quantification,and matrix effects were 0.02-5.5 μg/kg,0.06-10 μg/kg,and-98.8 to 13.9％(at 20 μg/kg),respectively.In five samples of each food matrix(chicken muscle,pork,beef,milk,and egg)purchased from large retailers in Seoul that were tested,none of the target analytes were detected.It has therefore been shown that this protocol is adaptable,accurate,and precise for the quantification of anthelmintic residues in foods of animal origin.

Residue analysis of tebufenozide and indoxacarb in chicken muscle, milk, egg and aquatic animal products using liquid chromatography-tandem mass spectrometry.

We developed an analytical method using liquid-liquid extraction (LLE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to detect and quantify tebufenozide (TEB) and indoxacarb (IND) residues in animal and aquatic products (chicken muscle, milk, egg, eel, flatfish, and shrimp). The target compounds were extracted using 1% acetic acid (0.1% acetic acid for egg only) in acetonitrile and purified using n-hexane. The analytes were separated on a Gemini-NX C18 column using (a) distilled water with 0.1% formic acid and 5 mm ammonium acetate and (b) methanol with 0.1% formic acid as the mobile phase. All six-point matrix-matched calibration curves showed good linearity with coefficients of determination (R2 ) ≥0.9864 over a concentration range of 5-50 µg/kg. Intra- and inter-day accuracy was expressed as the recovery rate at three spiking levels and ranged between 73.22 and 114.93% in all matrices, with a relative standard deviation (RSD, corresponding to precision) ≤13.87%. The limits of quantification (LOQ) of all target analytes ranged from 2 to 20 µg/kg, which were substantially lower than the maximum residue limits (MRLs) specified by the regulatory agencies of different countries. All samples were collected from different markets in Seoul, Republic of Korea, and tested negative for tebufenozide and indoxacarb residues. These results show that the method developed is robust and may be a promising tool to detect trace levels of the target analytes in animal products.

Development and validation of a solid-phase extraction method coupled with LC-MS/MS for the simultaneous determination of 16 antibiotic residues in duck meat.

The present study was carried out to determine 16 antibiotics belonging to seven different groups (tetracyclines, sulfonamides, penicillins, fluoroquinolones, macrolides, lincosamides and trimethoprims) in duck meat. A solid-phase extraction method based on Oasis HLB cartridges coupled with liquid chromatography-electrospray ionization tandem mass spectrometry was developed. Solutions of 0.1 m ethylenediaminetetraacetic acid disodium salt and 2% trifluoroacetic acid were used for the preliminary extraction of the target antibiotics from duck meat and n-hexane was used for purification prior to solid-phase extraction. Mobile phases composed of 0.1% trifluoroacetic acid in distilled water (solvent A) and 0.1% trifluoroacetic acid in methanol (solvent B), combined with a reversed-phase C18 analytical column, provided the optimal separation and signal intensity. The linearity of the method was assessed using six concentrations (5, 10, 20, 30, 40, and 50 µg/kg), and the recoveries, which were calculated at three spiking concentrations (5, 10 and 20 µg/kg), were in the range 69.8-103.3% with relative standard deviations (RSDs) ≤ 6.9% for the 16 tested antibiotics. Matrix effects ranging from -47.2 to -13.5% were observed for all the analytes, and the limits of quantitation (LOQ), which ranged from 4.93 to 26.21 µg/kg, were much lower than the maximum residue limits (MRLs) set by various regulatory authorities. Ten samples from a market were tested, and none of the target analytes were detected. Thus, a simple and versatile protocol has been developed to detect and quantify 16 antibiotics in duck meat samples.

Simultaneous determination of spinosad, temephos, and piperonyl butoxide in animal-derived foods using LC-MS/MS.

Pesticides, which are used as plant protection products, can enter the food chain, and exposure to these xenobiotics can cause a wide array of health problems in humans. Therefore, the objective of the present study was to develop an analytical method for the simultaneous determination of residual spinosad (sum of spinosyn A and D), temephos and piperonyl butoxide in porcine muscle, egg, milk, eel, flatfish and shrimp (sampling period: February to June 2018) using liquid chromatography-triple quadrupole tandem mass spectrometry (LC-MS/MS). The target analytes were extracted with a combination of acidified acetonitrile and ethyl acetate and subsequently purified with original QuEChERS kits (composed of magnesium sulfate and sodium chloride) as well as n-hexane. All analytes were separated on a reversed-phase analytical column using a mobile phase of (A) 0.1% formic acid containing 10 mm ammonium formate in distilled water and (B) methanol. Good linearity (R2 ≥ 0.980) was achieved over the tested concentration range (3.5-35 µg/kg for spinosyn A; 1.5-15 µg/kg for spinosyn D; 5-50 µg/kg for temephos and piperonyl butoxide) in matrix-matched standard calibrations. Fortified samples at three spiking levels yielded recoveries in the range of 71-105% with relative standard deviations ≤9.2%. The applicability of the method was evaluated via evaluating samples collected from a large wholesale market located in Seoul, and none of the samples contained any of the target analytes. In conclusion, the current approach is simple, efficient and reliable and can successfully determine the residual levels of spinosad, temephos and piperonyl butoxide in complex animal-derived food products.

Residual detection of naproxen, methyltestosterone and 17α-hydroxyprogesterone caproate in aquatic products by simple liquid-liquid extraction method coupled with liquid chromatography-tandem mass spectrometry.

In the present study, we aimed to develop a reliable screening method based on liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the detection and quantification of naproxen, methyltestosterone and 17α-hydroxyprogesterone caproate residues. The target analytes were extracted from samples of eel, flatfish and shrimp using acetonitrile with 1% acetic acid, followed by liquid-liquid purification with n-hexane. Chromatographic separation was achieved on a reversed-phase analytical column using 0.1% formic acid containing 10 mm ammonium formate in distilled water (A) and methanol (B) as mobile phases. All the matrix-matched calibration curves were linear (R2 ≥ 0.99) over the concentration range of the tested analytes. Recovery at three spiking levels (0.005, 0.01 and 0.02 mg/kg) ranged from 68 to 117% with intra- and inter-day precisions <10%. Five market samples for each matrix (eel, flatfish and shrimp) were collected and tested for method application. In summary, the proposed method is feasible to screen and quantify the analytes with high selectivity in aquatic food products meant for human consumption.

Quantitative determination of carbasalate calcium derived metabolites, acetylsalicylic acid and salicylic acid, in six animal foods using liquid-liquid extraction method coupled with liquid chromatography-tandem mass spectrometry.

This work describes a simple screening protocol for quantification of carbasalate calcium derived metabolites, acetylsalicylic acid (ASA) and salicylic acid (SA), in animal and aquatic food matrices using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The analytes were extracted from porcine muscle, milk, egg, shrimp, eel, and flatfish using acetonitrile, with the addition of formic acid as well as trifluoroacetic acid, followed by liquid-liquid purification with saturated n-hexane. A reverse-phase analytical column was employed with a mobile phase comprising (A) 1 mM ammonium acetate in distilled water and (B) methanol to achieve the best chromatographic separation. Matrix-matched calibration curves (R2 ≥ 0.9817) were constructed using six concentrations of 5, 10, 20, 30, 40, and 50 µg/kg in porcine muscle, milk, egg, shrimp, eel, and flatfish matrices. The calculated limits of quantification (LOQ) were 10 and 7 µg/kg, for ASA and SA, respectively. Recoveries of 67 to 102% with relative standard deviations (RSDs) of ≤9.0% (intra-day and inter-day) were obtained for all matrices at four spiking concentrations (5, 10, 20, and 50 µg/kg). The method was feasibly applied for monitoring market samples. In conclusion, the developed method is versatile, accurate, and precise for detecting and quantifying acetylsalicylic acid and salicylic acid residues in animal-derived foods meant for human consumption.

A modified QuEChERS method coupled with liquid chromatography-tandem mass spectrometry for the simultaneous detection and quantification of scopolamine, L-hyoscyamine, and sparteine residues in animal-derived food products.

We developed a modified Quick, Easy, Cheap, Effective, Rugged, and Safe (CEN QuEChERS) extraction method coupled with liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI+/MS-MS) to identify and quantify residues of three botanical alkaloids, namely, scopolamine, L-hyoscyamine, and sparteine, in animal-derived foods, including porcine muscle, egg, and milk. A combination of ethylenediaminetetraacetic acid disodium buffer and acetonitrile acidified with 0.5% trifluoroacetic acid was used as an extraction solvent, whereas QuEChERS (CEN, 15662) kits and sorbents were applied for cleanup procedures. The proposed method was validated by determining the limits of quantification (LOQs), with values of 1-5 µg/kg achieved for the target analytes in various matrices. Linearity was estimated from matrix-matched calibration curves constructed using six concentration levels ranging from 1- to 6-fold increases in the LOQs of each analyte, and the correlation coefficients (R2 ) were ≥0.9869. Recoveries (at three concentration levels of 1-, 2-, and 3-fold increases in the LOQ) of 73-104% were achieved with relative standard deviations (RSDs) ≤7.7% (intra-day and inter-day precision). Ten types of each matrix procured from large markets were evaluated, and all tested samples showed negative results. The current protocol is simple and versatile and can be used for routine detection of plant alkaloids in animal food products.

Quantification of artesunate and its metabolite, dihydroartemisinin, in animal products using liquid chromatography-tandem mass spectrometry.

An analytical approach using a modified quick, easy, cheap, effective, rugged, and safe extraction method followed by liquid chromatography with electrospray ionization tandem mass spectrometry was developed herein for the determination of artesunate and its metabolite, dihydroarteminsinin in porcine muscle, egg, eel, flatfish, and shrimp. 10% trichloroacetic acid in acetonitrile mixed with ethyl acetate was used as an extraction solvent. To obtain a good separation, a Phenomenex Kinetex reversed-phase analytical column was selected with mobile phase consisting of distilled water (A) and acetonitrile (B), both containing 0.05% formic acid. Good linearity was achieved using matrix-matched calibrations constructed from six concentrations (5-50 µg/kg) with determinant coefficients ≥0.9918. Recoveries estimated from three spiking concentrations (5, 10, and 20 µg/kg) ranged between 71.3 and 104.7% in all matrixes with relative standard deviations ≤8.3%. A variety of samples purchased from markets in Seoul were tested following the protocol described herein. The artesunate and dihydroarteminsinin were not detected in any matrix. The methodology proposed could be used for routine determination of artesunate and its metabolite, dihydroartemisinin in various animal products having variable percentages of fat and protein.

Determination of halquinol residual levels in animal-derived food products using liquid chromatography-tandem mass spectrometry.

A reliable and highly sensitive detection method based on liquid chromatography coupled with triple quadrupole electrospray tandem mass spectrometry (LC-MS/MS) analysis has been developed for determination and quantification of halquinol, including 5,7-dichloroquinolin-8-ol and 5-chloroquinolin-8-ol. The target analytes were extracted from porcine muscle, egg, milk, eel, flatfish and shrimp using a mixture of acetonitrile and ethyl acetate followed by liquid-liquid purification with n-hexane. The analytes were separated on an Agilent Eclipse XDB-C18 reversed-phase analytical column using 0.05% formic acid in distilled water and acetonitrile as mobile phases. Good linearity from six-point matrix-matched calibration was obtained with correlation coefficients (R2 ) ≥ 0.9904. Recoveries from three spiking levels (5, 10 and 20 µg/kg) ranged between 70.6 and 101.7% in various matrices with relative standard deviations ≤8.6%. Samples acquired from markets located in Seoul, Republic of Korea, tested negative for the target analytes. In conclusion, the proposed method is versatile and precise for the routine detection of halquinol residual levels in animal-derived food products intended for human consumption.

Determination of metoserpate, buquinolate, and diclofenac in pork, eggs, and milk using liquid chromatography-tandem mass spectrometry.

In this work, a method was developed for the simultaneous determination of residual metoserpate, buquinolate and diclofenac in pork, milk, and eggs. Samples were extracted with 0.1% formic acid in acetonitrile, defatted with n-hexane, and filtered prior to analysis using liquid chromatography-tandem mass spectrometry. The analytes were separated on a C18 column using 0.1% acetic acid and methanol as the mobile phase. The matrix-matched calibration curves showed good linearity over a concentration range of 5-50 ng/g with coefficients of determination (R2 ) ≥0.991. The intra- and inter-day accuracies (expressed as recovery percentage values) calculated using three spiking levels (5, 10, and 20 µg/kg) were 80-108.65 and 74.06-107.15%, respectively, and the precisions (expressed as relative standard deviation) were 2.86-13.67 and 0.05-11.74%, respectively, for the tested drugs determined in various matrices. The limits of quantification (1 and 2 µg/kg) were below the uniform residual level (0.01 mg/kg) set for compounds that have no specific maximum residue limit (MRL). The developed method was tested using market samples and none of the target analytes was detected in any of the samples. The validated method proved to be practicable for detection of the tested analytes in pork, milk, and eggs.

Simultaneous determination of clanobutin, dichlorvos, and naftazone in pork, beef, chicken, milk, and egg using liquid chromatography-tandem mass spectrometry.

A chromatographic method involving a single run was validated for the quantification of clanobutin, dichlorvos, and naftazone in products of animal origin. Pork, beef, chicken, milk, and egg samples were extracted with a solution of 0.1% formic acid in acetonitrile, defatted with n-hexane, centrifuged, and filtered prior to analysis using liquid chromatography-tandem mass spectrometry (LC-/MS/MS). The analytes were separated on a C18 column using a solution of 0.1% formic acid and 10 mM ammonium formate (A) and acetonitrile (B) as the mobile phase. A good linearity over 5-50 ng/g concentration range was obtained with coefficients of determination (R2) ≥ 0.9807. The intra- and interday accuracy (recovery %) calculated from 3 fortification levels (5, 10, and 20 ng/g) were 73.2-108.1% and 71.4-109.8%, and the precisions (expressed relative standard deviations (RSDs)) were 0.9-12.9% and 1.8-10.6%, respectively, for the 3 tested analytes in animal originated foods. The limits of quantification (LOQs) ranged between 0.1 and 1 ng/g, thus enabling the quantification of residual levels below the uniform maximum residue limit (MRL) of 0.01 mg/kg set for compounds having no MRL. The designated methodology was successfully applied to monitor various samples collected from Seoul; the tested analytes were not quantified in any of the market samples. Conclusively, the developed method is simple, sensitive, and accurate, and could be used for the detection of pharmaceuticals in various animal food matrices with variable protein and fat contents.

Development and validation of a simple solid-phase extraction method coupled with liquid chromatography-triple quadrupole tandem mass spectrometry for simultaneous determination of lincomycin, tylosin A and tylosin B in royal jelly.

We have developed an analytical method for the determination of lincomycin, tylosin A and tylosin B residues in royal jelly using liquid chromatography-triple quadrupole tandem mass spectrometry analysis. For extraction and purification, we employed 1% trifluoroacetic acid and 0.1 m Na2 EDTA solutions along with an Oasis HLB cartridge. The target antibiotics were well separated in a Kinetex EVO C18 reversed-phase analytical column using a combination of 0.1% formate acid in ultrapure water (A) and acetonitrile (B) as the mobile phase. Good linearity was achieved over the tested concentration range (5-50 µg/kg) in matrix-matched standard calibration. The coefficients of determination (R2 ) were 0.9933, 0.9933 and 0.996, for tylosin A, tylosin B and lincomycin, respectively. Fortified royal jelly spiked with three different concentrations of the tested antibiotics (5, 10 and 20 µg/kg) yielded recoveries in the range 80.94-109.26% with relative standard deviations ≤4%. The proposed method was applied to monitor 11 brand of royal jelly collected from domestic markets and an imported brand from New Zealand; all the samples tested negative for lincomycin, tylosin A and tylosin B residues. In conclusion, 1% trifluoroacetic acid and 0.1 m Na2 EDTA aqueous solvents combined with solid-phase extraction could effectively complete the sample preparation process for royal jelly before analysis. The developed approach can be applied for a routine analysis of lincomycin, tylosin A and tylosin B residues in royal jelly.

Development and validation of modified QuEChERS method coupled with LC-MS/MS for simultaneous determination of cymiazole, fipronil, coumaphos, fluvalinate, amitraz, and its metabolite in various types of honey and royal jelly.

Over the past few decades, honey products have been polluted by different contaminants, such as pesticides, which are widely applied in agriculture. In this work, a modified EN - quick, easy, cheap, effective, rugged, and safe (QuEChERS) extraction method was developed for the simultaneous quantification of pesticide residues, including cymiazole, fipronil, coumaphos, fluvalinate, amitraz, and its metabolite 2,4-dimethylaniline (2,4-DMA), in four types of honey (acacia, wild, chestnut, and manuka) and royal jelly. Samples were buffered with 0.2M dibasic sodium phosphate (pH 9), and subsequently, acetonitrile was employed as the extraction solvent. A combination of primary secondary amine (PSA) and C18 sorbents was used for purification prior to liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI+/MS-MS) analysis. The estimated linearity measured at six concentration levels presented good correlation coefficients (R2)≥0.99. The recovery, calculated from three different spiking levels, was 62.06-108.79% in honey and 67.58-106.34% in royal jelly, with an RSD<12% for all the tested compounds. The matrix effect was also evaluated, and most of the analytes presented signal enhancement. The limits of quantification (LOQ) ranged between 0.001 and 0.005mg/kg in various samples. These are considerably lower than the maximum residue limits (MRL) set by various regulatory authorities. A total of 43 market (domestic and imported) samples were assayed for method application. Among the tested samples, three samples were tested positive (i.e. detected and quantified) only for cymiazole residues. The residues in the rest of the samples were detected but not quantified. We concluded that the protocol developed in this work is simple and versatile for the routine quantification of cymiazole, 2,4-DMA, fipronil, coumaphos, amitraz, and fluvalinate in various types of honey and royal jelly.