A collaborative study to establish the national standard for SARS-CoV-2 RNA nucleic acid amplification techniques (NAAT) in Taiwan.

The conventional PCR remains a valuable method to detect the newly emergent coronavirus rapidly and accurately. Our investigation aimed to establish the standard materials of SARS-CoV-2 for NAAT detection. We provided formalin-inactivated SARS-CoV-2 and confirmed RNA copy numbers. In addition, the virus genome was confirmed with whole-genome sequencing and identified as Wuhan/WI04/2019. Seven laboratories were invited for this collaborative study, according to the reporting data, we determined the SARS-CoV-2 with the unit of 6.35 Log10 copies/mL as the national standard. The availability of the national standard (NS) of SARS-CoV-2 will facilitate the standardization and harmonization of SARS-CoV-2 NAAT assays.

Establishment of an animal challenge model as a potency assay for an inactivated Enterovirus Type 71 vaccine.

Enterovirus 71 (EV71) belongs to the Enterovirus genus of the Picornaviridae family, and its occurrence in Asia is associated with hand-foot-and-mouth disease (HFMD), leading to death in some cases, in young children. An effective EV71 vaccine is therefore urgently needed. In this study, we established a two-step EV71 vaccine potency model. Intraperitoneal injections in 2-day-old suckling mice were used to establish the LD50 of EV71 B4, B5, C2, C4, and C5 subgenotypes. Only C4 caused hind limb paralysis in mice (LD50: 2.62 ± 0.45). EV71 VP1 protein was identified in the brain tissues at histology. In the second phase of the model, 3-week-old female ICR mice received one primary and two boosting i.p. injections of formalin-inactivated EV71 B4 and C4 vaccine. Immunized serum was neutralized in vitro with EV71 C4 and applied to the murine challenge model. The C4 vaccine-immunized serum exhibited the highest protective titre (ED50 = 114.6), while the B4 immunized serum had the weakest protective titre (ED50 = 34.3). Additionally, human plasma and intravenous immunoglobulin displayed significant protection in the neutralization assay. Our results could facilitate candidate EV71 vaccine immunogenicity and efficacy evaluations, and may help establish reference EV71 antisera in the future.

Analyzing titers of antibodies against bacterial and viral antigens, and bacterial toxoids in the intravenous immunoglobulins utilized in Taiwan.

Intravenous immunoglobulin (IVIG) manufactured from human plasma contains IgG as the primary ingredient, and is used for indications such as immunodeficiency syndrome. Available IVIGs in Taiwan are either manufactured from Taiwanese or North American plasma. The effectiveness of the national immunization program of Taiwan can be evaluated by analyzing and comparing IVIG antibody titers that are induced through the corresponding vaccines (tetanus, diphtheria, and pertussis, measles, rubella, hepatitis A, hepatitis B and varicella). Both enzyme-linked immunosorbent assay (ELISA) and the in vitro neutralization test demonstrated that all IVIGs provide adequate clinical protection against diphtheria and tetanus toxins. ELISA results further revealed that plasma of Taiwanese subjects contains higher levels of pertussis toxin and filamentous hemagglutinin antibodies, when compared to foreign IVIGs. This may be related to the later adoption of acellular pertussis vaccine in Taiwan. Antibodies titers against measles, rubella, hepatitis A, and varicella-zoster virus were otherwise low. Low titers of hepatitis B surface antigen antibodies are present in Taiwanese plasma IVIG, indicating immune memory decline or loss. In conclusion, our results show that Taiwanese IVIG contains varying titers of vaccine-induced antibodies, and serves as a guide for future amendments to Taiwan's immunization program.

Neutralization of five subgenotypes of Enterovirus 71 by Taiwanese human plasma and Taiwanese plasma derived intravenous immunoglobulin.

Enterovirus 71 (EV71) commonly occurs in children, causing hand, foot and mouth disease (HFMD) in about 29% of patients. Studies have suggested that patients develop meningitis and encephalopathy with a mortality rate of 4-26%. EV71 subgenotypes including B4, B5, C2, C4 and C5 have caused HFMD epidemics in Taiwan. In terms of therapeutical strategy, intravenous immunoglobulin (IVIG) has been shown to improve patient conditions. In this study, the EV71 neutralizing titer was evaluated in 75 human plasmas and 8 lots of Taiwanese plasma derived IVIG. Results showed that human plasmas and IVIG significantly neutralized B4 and C2 subgenotypes. Four percent of human plasma contained neutralizing antibody titer of 1:128 against B4 and C2. Most IVIG lots possessed a median effective dose of over 100 against B4 and C2. IVIG lots had an average neutralizing capacity of 5.60, 0.90, 4.30, 1.12 and 0.77 log10 CCID50/ml against B4, B5, C2, C4 and C5, respectively. In conclusion, effective neutralization of B4 and C2 could be due to their earlier appearance in the EV71 epidemiology timeline of Taiwan. IVIG derived from Taiwanese plasma may be desirable for treatment of patients infected with EV71 of specific subgenotypes.

Collaborative study to establish a proficiency panel of hepatitis B surface antigens for evaluating in vitro diagnostics.

Hepatitis B virus (HBV) infection is a critical health issue. In hospitals, in vitro diagnostic devices (IVDs) are used to detect HBV infections. The Taiwan Food and Drug Administration has prepared a candidate hepatitis B surface antigen (HBsAg) proficiency panel as an analytical standard to evaluate the sensitivity of the postmarketing surveillance of HBV IVDs. The sensitivity and stability of the proficiency panel, which comprises 5 HBsAg positive sera (SN-A to E) and 1 negative serum, was determined by a collaborative study involving 8 collaboratories. The potency of the candidate proficiency panel was consistent among all collaboratories. The potencies of sera SN-A to E in semiquantitative and quantitative analyses were 2.823-0.011 and 2.229-0.009 IU/mL, respectively. The mean coefficients of variation (CV) of intra and interday precision among the collaboratories were <7%. Accelerated degradation and long-term storage tests were used as stability assays. The candidate HBsAg proficiency panel exhibited good stability under different storage conditions, while all CVs were <6%. In conclusion, this candidate HBsAg proficiency panel is credible and reliable. Therefore, it can be used for postmarketing surveillance of HBV IVDs and quality assessment by clinical researchers and IVD manufacturers.

Stable carbon isotopic characterization of rice vinegar protein as an intrinsic reference for discriminating the authenticity of brewed rice vinegar.

Rice vinegar plays an important role in daily life. However, some unscrupulous manufacturers may deliberately add synthetic acetic acid in vinegar products to reduce fermentation time and save production costs. To protect the rights and health of consumers, vinegar authenticity must be controlled. The rice vinegar protein was used as an intrinsic reference and its stable carbon isotope ratio (δ13Cprotein) was analyzed by elemental analyzer-isotope ratio mass spectrometry. The stable carbon isotope ratio difference between the acetic acid and the rice vinegar protein (Δδ13Cacetic acid-protein) was calculated to evaluate vinegar authenticity. Sixteen rice vinegar samples were analyzed and a stable carbon isotopic pattern of rice vinegar was established by the 95% confidence interval for Δδ13Cacetic acid-protein (0.27‰-2.10‰). An acetic acid adulteration curve of Δδ13Cacetic acid-protein was also assumed according to the data from rice vinegar samples, and its validity was confirmed by rice vinegar deliberately blended with acetic acid at different ratios (25, 50, and 75%). The Δδ13Cacetic acid-protein values of the adulterated vinegars decreased with increasing amounts blended acetic acid, but the δ13Cprotein values did not, showing that rice vinegar protein could be used as an intrinsic reference for identifying the adulterated rice vinegar. The rice vinegar adulterated with acetic acid at higher than approximately 10% could be detected.

Inorganic arsenic speciation analysis in food using HPLC/ICP-MS: Method development and validation.

Arsenic (As) compounds can be classified as organic or inorganic, with inorganic arsenic (iAs) having significantly higher toxicity than organic As. As may accumulate in food materials that have been exposed to As-contaminated environments. Thus, the "Sanitation Standard for Contaminants and Toxins in Foods" published by the Ministry of Health and Welfare set the standard limits for iAs content in rice, seaweed, seafood, and marine oils to safeguard public health. Therefore, a robust analytical method must be developed to selectively and quantitatively determine iAs content in rice, seaweed, seafood, and marine oils. Herein, we reported and verified the method of combined high-performance liquid chromatography/inductively coupled plasma-mass spectrometry (HPLC/ICP-MS) to determine iAs content in a wide variety of food. The fish oil samples were spiked with different concentrations of the As(III) standard solution, and their iAs analyzes were obtained via extraction procedures using the 1% (w/w) nitric acid (HNO3) solution containing 0.2 M hydrogen peroxide (H2O2) under sonication. The extracts were subsequently analyzed for their As(V) contents using HPLC/ICP-MS with aqueous ammonium carbonate as the mobile phase. The As(III) species had completely oxidized into the As(V) species, which prevented interferences between organic and iAs during chromatography. The method showed good extraction efficiencies (generally >90%) for the iAs samples, and their limits of quantification in fish oil were 0.02 mg/kg. The method was verified via the iAs speciation analytes of rice, seaweed, seafood, and marine oil matrices. The average recoveries for the fortified samples of each matrix ranged from 87.5 to 112.4%, with their coefficients of variation being less than 10%. Surveillance studies were conducted on the iAs contents of food samples purchased from local Taiwanese markets. The results showed that the only Hijiki (Sargassum fusiforme) higher than the maximum limit of the sanitation standard for iAs in seaweed, whereas the remaining samples met their corresponding requirements. This method is quick and straightforward, and it can be applied for the routine analysis of iAs content in a wide variety of food products to ensure public health safety.

Evaluation and optimization of a HS-SPME-assisted GC-MS/MS method for monitoring nitrosamine impurities in diverse pharmaceuticals.

The probable carcinogenic nitrosamine impurities, such as N-nitrosodiethylamine (NDEA) and N-nitrosodimethylamine (NDMA), have been detected from various pharmaceuticals in recent years. The sensitive chromatographic methods, including liquid chromatography (LC) and gas chromatography (GC), have been applied for analyzing nitrosamines in the pharmaceutical substrates, such as sartans, ranitidine and metformin. In comparison of LC, the efficacy of GC for analyzing multiple nitrosamines in diverse pharmaceuticals will be limited or attenuated owing to the chemical properties of target analytes or matrix hinderance of pharmaceutical substrates. To extend the applicability of GC analysis for multiple nitrosamines in pharmaceuticals, this study presented a gas chromatograph tandem mass (GC-MS/MS) method for monitoring 14 nitrosamines within 44 pharmaceuticals, whereas the headspace-solid phase microextraction (HS-SPME) sampling mode was introduced. Chromatographic separation was achieved on a DB-heavyWax column (30 m × 0.25 mm; i.d., 0.25 µm), whereas the HS-SPME sampling mode with a 50/30 µm DVB/CAR/PDMS extracting fiber was applied for comparison of the direct injection mode. Meanwhile, the HS-SPME conditions were optimized to evaluate the effects of the parameters on analyzing total nitrosamines in pharmaceuticals by GC-MS/MS. The optimal conditions of HS-SPME were as follows: extracting solution of 90% NaCl, HS incubation time 1 min, SPME adsorbing at 80 â„ƒ for 30 min, and desorbing at 250 â„ƒ for 5 min. The limit of quantification (LOQ) for 14 nitrosamines in pharmaceutical matrices under the optimal conditions was 0.05 µg/g for the optimal HS-SPME, whereas the value was 0.05-0.25 µg/g for direct injection.

The growth-promoting effect of KGF on limbal epithelial cells is mediated by upregulation of DeltaNp63alpha through the p38 pathway.

Corneal epithelial stem cells are thought to reside in the limbus, the transition zoon between cornea and conjunctiva. Keratinocyte growth factor (KGF) and hepatocyte growth factor (HGF) are two paracrine factors that regulate the proliferation, migration and differentiation of the limbal epithelial cells; however, the underlying mechanisms are still poorly understood. In an ex vivo limbal explant culture, we found that KGF is a more potent growth stimulator for the epithelial outgrowth than HGF. Immunofluorescence studies of the epithelial outgrowth from cells treated with HGF or KGF showed similar expression patterns of keratin-3 and keratin-14. Interestingly, p63 was highly expressed in KGF-treated limbal epithelial sheets but not in those treated with HGF. Kinase inhibitor studies showed that induction of DeltaNp63alpha expression by KGF is mediated via the p38 pathway. The effect of KGF on limbal epithelial outgrowth was significantly reduced when endogenous DeltaNp63alpha was suppressed, suggesting that KGF-induced limbal epithelial outgrowth is dependent on the expression of DeltaNp63alpha. Our findings strongly suggest that limbal keratocytes regulate limbal epithelial cell growth and differentiation through a KGF paracrine loop, with DeltaNp63alpha expression as one of the downstream targets.

Quantitative analysis of fragrance allergens in various matrixes of cosmetics by liquid-liquid extraction and GC-MS.

Fragrances are the most common chemicals in cosmetics to which people expose every day. However, the unwanted allergic reactions such as contact dermatitis caused by direct contact with fragrances may happen. In Directive 2003/15/EC of the EU, cosmetic product containing one or more of 26 fragrance allergens must be declared on the package label. In addition, commission regulation (EU) 2017/1410 amending Annexes II and III of cosmetic regulation 1223/2009 restricted fragrance chemical of methyl eugenol, and prohibited Lyral, atranol, chloroatranol to be used in cosmetic. In this study, an efficient and sensitive GC-MS method for 3 banned fragrances, 26 fragrance allergens along with restricted methyl eugenol in cosmetics was established. Sample preparation by liquid-liquid extraction was developed by testing various solvent systems to simplify traditional complex extraction methodologies. Validation of the proposed method showed good linearities in a wide concentration ranges of 0.1-10 µg/mL. The intra-day and inter-day recoveries were between 84.4 and 119% with coefficient of variation (CV) below 13.5%. The limit of quantifications (LOQs) of 27 fragrance allergens were in the range of 2-20 µg/g. A surveillance study consisted with 82 cosmetics was conducted, among which 31 products claimed fragrance-free. The results showed some fragrance-free claims were false. In the other hand, there were seven cosmetics labeled containing Lyral, but only four were detected. The top fragrance allergens detected in the samples were linalool, limonene, and geraniol. The analysis of fragrance allergens in cosmetics indicated that potential contact allergy related to these products should be considered, even though some fragrance allergens were from natural extracts, such as oak moss absolute.

Comprehensive detection of 120 additives in food using nontargeted MS data acquisition.

The compliance assessment on the labeling of food additives is a hard job, because there are nearly thousand legal food additives can be used in food, and countless illegal additives must also deal with. This study developed a non-targeted data acquisition screening method based on liquid chromatography high resolution mass spectrometry (HRMS) in which a precursor ion and two product ions of each analyte are able to be recorded. The high throughput screening method worked as foodomics that characterized and identified every food components as long as they were ionized in terms of theory. The data acquisition method called data independent acquisition (DIA) was achieved by a full scan form m/z 70-1050, and then followed wide window fragmentations of product ions recording. A full scan and the followed fragmentations generated 21 spectra in 2.6 s contributed about 6 data points for a typical 0.2-0.3 min width peak in HPLC. A detection database list of 120 additives included 79 colorants, 13 sweeteners, 12 preservatives and 7 antioxidants was established. Thirty-three commercial samples including beverages, candies, and sauces were surveyed for testing additives. Sweeteners (rebaudioside A) and flavoring agents (malic acid and fumaric acid) were found the most under declared additives. HPLC column often do not provide adequate retention for highly polar compounds such as organic acids (flavoring agents). In this study they were coeluted, but were able to be separated and determined by HRMS worked as the secondary separation tool. The surveillance results showed there is still room for food manufacturers to improve the connection between their product information and consumers.

Determination of cannabinoids in hemp oil based cosmetic products by LC-tandem MS.

Cosmetic products containing hemp seed oil as permitted raw materials required the specific compound delta-9-tetrahydrocannabinol (THC) below 10 µg/g. THC was the main psychoactive constituent of cannabis. Since hemp seed oil became an increasingly popular ingredient in cosmetics over the last few years, an efficient and reliable analytical method for THC and other cannabinoids in cannabis-infused cosmetic products was in need. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of delta-9-tetrahydrocannabinol (THC), cannabidiol (CBD), and cannabinol (CBN) in hemp seed oil based cosmetic products was developed. Method validation was performed by fertilizing blank samples with analytes and internal standards (THC-d3, CBD-d3, and CBN-d3). Chromatographic method utilized a Xbridge BEH Shield RP18 column with gradient elution containing 10 mM ammonium formate in water and methanol provided successful separation of THC, CBD, and CBN in cosmetic matrix. The combination of MS detection in positive electrospray ionization (ESI) and multiple reaction monitoring (MRM) mode offered rapid run time 13 minutes with limit of quantification (LOQ) of 0.05 µg/g. The intra- and inter-day recoveries were 79.23-114.04% and 83.55-111.61% with spiking levels ranged between 0.05 µg/g and 0.5 µg/g, respectively. Surveillance results of 90 cosmetic products showed 22, 34, and 5 products containing THC (0.06-1777 µg/g), CBD (0.47-37217 µg/g), and CBN (2.2-25.2 µg/g), respectively. This validated method offered accurate, reliable, and fast way for the determination of drug contaminations including THC, CBD, and CBN in cosmetics. The surveillance results for commercial cosmetic products purchased in Taiwan between 2018-2020 provided valuable background references for THC, CBD, and CBN in hemp seed oil based cosmetic products, and could be used for administration purpose.

Elucidation of two new corticosteroids, betamethasone dibutyrate and betamethasone tributyrate.

Corticosteroids were used normally as anti-inflammatory drugs. However, in some area certain corticosteroids might be illegally used as growth promoting agent in feed, and as prohibited doping substances in game and sport for human or/and animal performance-enhancing. Synthesized structural similar corticosteroids were popular in black market because they can pass routine drug screening. In this study two new artificial synthesized corticosteroids were found in claimed hydrolyzed wheat product. Liquid chromatography coupled with high resolution mass spectrometry (HRMS, Orbitrap) was applied to separate and elucidate the corticosteroids in the sample. Two unknown peaks with optical spectra similar to corticosteroids were first screened out at the beginning, and then their accurate molecular weight (M + H+) m/z 533.29059 and m/z 603.33289 were detected by HRMS. Element formulas of unknowns were calculated by the accurate mass and isotopes abundance. Structures were proposed by their fragment ions at high energy collision dissociation (HCD, 10 eV) and compared with candidate standard compounds. The two unknowns shared similar molecular skeleton with steroid core structure and presented man made fluorine element in their molecule. As the results, the unknowns in the sample were artificial synthesized, and the sample product was not a real food. The detected corticosteroids were also synthesized as reference compounds for conformation. Two new corticosteroids named betamethasone dibutyrate and betamethasone tributyrate were found and first time reported in this work. The legality of structural similar/modified corticosteroids were blurry and their safety were unverified. The confirmed identifications of two new found corticosteroids, and their mass spectra were provided in this paper for the reference of drug detection.

Development of the species-specific multiplex PCR and DNA sequencing methods for rapid authentication of Isatidis Folium and its adulterants.

Isatis indigotica Fort. (family Cruciferae), is an herb widely used in traditional herbal medicine and its dried leave was named "ISATIDIS FOLIUM". Baphicacanthus cusia (Ness) Bremek. and Polygonum tinctorium Ait. are commonly misused as ISATIDIS FOLIUM in Chinese Medicine pharmacy. For the purpose of being not misused, specific primers based on the sequence difference of chloroplast trnH-psbA intergenic spacer were designed and multiplex polymerase chain reaction method (multiplex PCR) was developed. In this study, 29 original herbal materials were analyzed and our results show that DNA size after multiplex PCR was able to distinguish variations between three herbs. DNA fragments of 464, 297, 170 base pairs (bps) were represented for I. indigotica and B. cusia and P. tinctorium, respectively. In conclusion, our investigations demonstrate that molecular identification method provides more accurate results for medicinal plants detection and good quality control of ISATIDIS FOLIUM.

Simultaneous LC-MS/MS screening for multiple phenethylamine-type conventional drugs and new psychoactive substances in urine.

New psychoactive substances are being launched in the drug market at a rapidly growing pace. More than 950 new psychoactive substances have been reported to the United Nations Office on Drugs and Crime. The development of new psychoactive substance abuse has drawn risks on public health and safety. Phenethylamines, along with other stimulants, accounted for the majority of the new psychoactive substances being reported in the past decade. This study presents a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous screening of 74 conventional and artificial phenethylamines in urine samples. The chromatographic analysis was performed by a direct dilute-and-shoot procedure using a Phenomenex Kinetex® Phenyl-Hexyl column (10 cm × 2.1 mm i.d., 1.7 µm) and two mobile phases (A: 0.1% formic acid aqueous solution with 5 mM ammonium acetate, B: 0.1% formic acid methanolic solution). The mass fragments were collected under the multiple reaction monitoring mode. The linearity range located in 1.0-50.0 ng/mL for quantitative analysis. The limit of detection and lower limit of quantification for 74 phenethylamines were 0.5 ng/mL and 1.0 ng/mL, respectively. The method was validated and further applied to analyze authentic urine samples. Twenty samples were tested positive of seven phenethylamines from 67 samples, whereas the contents detected were 9.8 ng/mL to 147.1 µg/mL with dilution factors of 40 to 20,000 folds.

Simultaneous Analysis of the Stable Carbon Isotope Ratios of Acetoin and Acetic Acid by GC-C-IRMS for Adulteration Detection in Brewed Rice Vinegar Products.

In this study, we developed a method to simultaneously measure the stable carbon isotope ratio for acetic acid (δ 13Cacetic acid) and acetoin (δ13Cacetoin) in rice vinegar by gas chromatography-combustion-isotope ratio mass spectrometry. The method showed good precision and accuracy. With this method, data from 16 brewed rice vinegars and 10 acetic acid samples were used to evaluate the feasibility of adulteration detection. On the basis that all δ13Cacetoin values of brewed rice vinegars are nearly constant, a characteristic pattern of the stable carbon isotope in rice vinegar was built with the 95% confidence intervals for δ13Cacetic acid (-26.97 to -25.38‰), δ13Cacetoin (-28.14 to -27.09‰), and Δδ13C (0.61 to 2.27‰). An adulteration detection curve of Δδ13C was proposed based on the results of vinegar and acetic acid samples and confirmed by vinegar spiked with different amounts of acetic acid. This method could be useful in estimating the blending ratio of adulterated rice vinegar products. Products containing more than 10% of synthetic acetic acid could be possibly identified.

A multi-analyte LC-MS/MS method for screening and quantification of nitrosamines in sartans.

An incident of sartan medicine contamination was notified by Europe in June 2018. The contaminant was identified as a probable carcinogenic nitrosamine and the recalls of sartan medicines were soon made. Since then, more nitrosamine contaminants in sartan medicines were reported. To broaden the applicability and variety in nitrosamine determination, a multi-analyte method is required. In this study, a feasible and sensitive multi-analyte LC-MS/MS method for determination of 12 nitrosamines in sartans was established, where the active pharmaceutical ingredients and final products merchandised in Taiwan were also examined. Chromatographic separation was achieved on an Xselect® HSS T3 column (15 cm × 3 mm i.d., 3.5 µm) with gradient elution using mobile phase A consisting of 0.1% formic acid in water and mobile phase B consisting of 0.1% formic acid in acetonitrile/methanol (2:8). Validation of the proposed method was also carried out. The limit of detection and limit of quantification for 12 nitrosamines were 20 ng/g and 50 ng/g, respectively. The intra-day and inter-day recoveries of nitrosamines were among 80-120% with precision of 20% for most nitrosamines within sartans matrices. The method was successfully established and applied to authentic samples which a total of 98 positive samples containing 5 distinct nitrosamines, including N-nitrosodiethylamine, N-nitrosodimethylamine, N-nitroso-N-methyl-4-aminobutyric acid, N-nitrosomorpholine and N-nitrosopiperidine, were detected from 557 authentic samples.

A LC-MS/MS method for determination of 73 synthetic cathinones and related metabolites in urine.

Synthetic cathinones, which are a group of ß-keto analogs of phenethylamine, have been reported as the most emerging new psychoactive substances in the past decade. The quantity and variety of synthetic cathinones have continued to increase, which poses considerable risks to public health and social security. In this study, an analytical method based on liquid chromatography-tandem mass spectrometry (LCMS/MS) was established for the simultaneous determination of 73 synthetic cathinones and related metabolites in urine. The chromatographic analysis was performed using a Kinetex® Biphenyl column (10 cm ×2.1 mm, 1.7 µm), applying a gradient mobile phase, comprising 0.1 % formic acid aqueous solution with 5 mM ammonium acetate and 0.1 % formic acid methanolic solution; the entire run time of the analysis was within 8 min. The multiple reaction monitoring (MRM) mode was employed to collect the monitoring and quantitative ion pairs. Intra-day/inter-day precision and accuracy were less than 10 % for all the studied analytes. The limits of detection and quantification for all the analytes were 0.1-0.5 ng/mL and 0.5-1.0 ng/mL, respectively. The matrix effect was satisfactory for all the analytes, with a deviation lower than 20 %. The present method was further applied to 67 authentic urine samples in which 13 different synthetic cathinones were detected from 32 positive samples. The abuse of poly-synthetic cathinones was examined that up to seven items was detected in one case from authentic samples in this study.

Determination of polycyclic aromatic hydrocarbons (PAHs) in cosmetic products by gas chromatography-tandem mass spectrometry.

A gas chromatography coupled with tandem mass spectrometry (GC-MS/MS) method is developed to determine 18 representative polycyclic aromatic hydrocarbons (PAHs) in cosmetics, including Benzo[a]pyrene (BaP) and others. The method offers high sensitivity and selectivity under selected reaction monitoring (SRM) mode to satisfy the requirements of both quantitation and qualitation. The extraction solvent system used in this study is acetone/hexane 1:1 (v/v) and other purification procedure is unnecessary. The linearities of 18 PAHs are validated in different concentration in the range of 0.25-20 ng/mL individually with coefficient correlation (r) higher than 0.996. The recoveries for spiking 3 different concentrations are from 87.40% to 120.44% for 18 PAHs and the coefficient of variation (CV) are below 12.32%. Limit of quantification (LOQ) of 18 PAHs is in the range of 0.05-0.2 mg/kg. A matrix enhancement effect is observed and can be compensated with deuterated internal standard. The method has been successfully applied to 73 samples, over 40 of them are lipsticks. The results show none of the samples detect Benzo[a]pyrene (BaP) and Dibenzo[a,h]anthracene (DBA), both are classified as the most carcinogenic. 8 PAHs are detected and the average value between 0.08 and 0.27 mg/kg. This study offers a sensitive and simple method to analyze 18 representative PAHs successfully and can be applied to cosmetic products and raw materials.

Distribution of antibiotic resistance genes among Staphylococcus species isolated from ready-to-eat foods.

We investigated antibiotic resistance of staphylococci isolated from 1128 samples of high-circulating RTE foods in Taiwan. A total of 111 Staphylococcus aureus and 709 coagulase-negative staphylococci (CoNS) comprising 23 species were isolated. The prevalence of S. aureus differed in various category of RTE foods, highest in fresh-cut fruits/vegetables (20.5%) and lowest in low-water activity (LWA) foods (0.7%). The overall staphylococcal contamination was highest in fresh-cut fruits/vegetables (62.2%), in which multiple isolates (up to 10) or species (up to 6) in single sample were frequently found. Distinct distribution of species contributed to unique feature in each category. Prevalence of antibiotic-resistant S. aureus was higher in fresh-cut fruits/vegetables samples (14.2% in 127) compared to other food categories (0-7.1%). A total of 4 MRSA carrying SCCmec type IV or VT were identified (3.6% in 111), in which 3 belonged to sequence type ST59 and one was ST5. Among CoNS, S. epidermidis and S. warneri exhibited higher non-intrinsic antibiotic resistance than other species. Of 41 methicillin-resistant CoNS (5.8% in 709) isolates, SCCmec type IV (n = 16) and type VT (n = 6) were most frequent. Isolates of S. saprophyticus, S. xylosus and S. sciuri displayed high rates of resistance to fusidic acid. Novel fusB-family determinants were identified in S. xylosus, S. sciuri and S. kloosii, which may contribute to their intrinsic resistance to fusidic acid. Compared to other food categories, fresh-cut fruits/vegetables were more contaminated by staphylococci carrying non-intrinsic resistance determinants including methicillin resistance. This nation-wide study demonstrated that some categories may have potential risk for transmitting antibiotic resistance, in which S. epidermidis and S. warneri should be gotten more attention.