Liquid chromatography-quadrupole orbitrap and liquid chromatography-tandem mass spectrometry system for rapid identification and quantitation of thirty nonsteroidal anti-inflammatory drugs and acetaminophen in illegal products.

RATIONALE: As the public interest in healthcare increases, illegal dietary supplements, foods, and drugs containing unauthorized pharmaceutical ingredients, including nonsteroidal anti-inflammatory drugs (NSAIDs) and acetaminophen, have been identified. Excessive and unintentional consumption is toxic to the gastrointestinal tract, kidneys, and liver; therefore, these pharmaceuticals must be monitored using analytical methods. METHODS: A rapid and reliable analysis system involving liquid chromatography-quadrupole orbitrap mass spectrometry (LC-Q-Orbitrap/MS) and liquid chromatography-tandem mass spectrometry (LC/MS/MS) was established and validated to identify and quantify 30 NSAIDs and acetaminophen. In addition, we obtained the MS2 spectrum for each component with the proposed structure of the fragment ions. RESULTS: The analytical method was applied to 505 samples of illicitly distributed dietary supplements, foods, and pharmaceuticals. Non-steroidal analgesics were detected in 126 samples. Carbamazepine (42.9%) and diclofenac (30.2%) were the most detected components in the samples; other pharmaceutical adulterants were also detected in some cases. Additionally, we present the identification of an unknown component, dexamethasone (799 µg/g), using LC-Q-Orbitrap/MS in a sample containing the unknown component with meloxicam (15.4 mg/g). CONCLUSIONS: The developed analysis system, consisting of qualitative analysis using LC-Q-Orbitrap/MS and quantitative analysis using LC/MS/MS, can rapidly and accurately identify and quantify NSAIDs and acetaminophen while also identifying non-analytical components.

Novel OBI noise reduction technique by using similar-OBI estimation in optical multiple access uplink.

A novel optical beat interference (OBI) noise reduction technique for optical multiple access uplink using the same wavelength is presented. The OBI noise is estimated by similar-OBI and mitigated by subtracting estimated OBI noise using the digital signal processing technique. To derive similar-OBI, an RF clipping tone is transmitted out of signal band. We numerically and experimentally verified the validity of proposed OBI noise reduction method in orthogonal frequency division multiplexing access (OFDMA) passive optical network (PON) uplink where the OBI noise has been reduced and signal to noise ratio (SNR) has been improved from 4.4 dB to 10.6 dB.

Mitigation of timing offset effect in IM/DD based OFDMA-PON uplink multiple access.

In orthogonal frequency division multiple access based passive optical network (OFDMA-PON) uplink, synchronization between optical network units (ONUs) is very important to maintain orthogonality. The synchronization among uplink signals is considered as one of the main challenges in OFDMA-PON due to optical path difference. In this paper, the performance degradation according to timing offset between ONUs is experimentally analyzed. And we propose and demonstrate timing offset effect reduction in asynchronous multiple access by using CP extension and filter bank based multicarrier (FBMC) system in intensity modulation/direct detection (IM/DD) based OFDMA-PON uplink transmission.

Dynamic methylation pattern of the methyltransferase1o (Dnmt1o) 5'-flanking region during mouse oogenesis and spermatogenesis.

DNA methyltransferase1o (Dnmt1o), which is specific to oocyte and preimplantation embryo, plays a role in maintaining DNA methylation in mammalian cells. Here, we investigated the methylation status of CpGs sites in the Dnmt1o 5'-flanking region in germ cells at different stages of oogenesis or spermatogenesis. The methylation levels of the CpG sites at the 5'-flanking regions were hypermethylated in growing oocytes of all follicular stages, while the oocytes in meiotic metaphase II (MII) were demethylated. The methylation pattern within the CpGs sites in the 5'-flanking region, however, was dramatically changed during spermatogenesis. We observed that there was significant non-CpG methylation both in MII oocytes and spermatocytes. Although a low methylation level in non-CpG sites was observed in primary and secondary oocytes, the CpA site of position 25 and CpT site of position 29 within the no-CpG region in the 5'-flanking region of Dnmt1o was highly methylated in MII oocytes. During spermatogenesis, the low degree of methylation at CpG sites in spermatocytes increased to a higher degree in sperm, while the high ratio of methylation in non-CpG sites in spermatocytes decreased. Together, germ cells showed inverted methylation patterns between CpG and non-CpG sites in the Dnmt1o 5'-upstream region, and the methylation pattern during oogenesis did not drastically change, remaining generally hypomethylated at the MII stage.

Centipede grass exerts anti-adipogenic activity through inhibition of C/EBPß, C/EBPα, and PPARγ expression and the AKT signaling pathway in 3T3-L1 adipocytes.

BACKGROUND: Centipede grass (CG) originates from China and South America and is reported to contain several C-glycosyl flavones and phenolic constituents, including maysin and luteolin derivatives. This study aimed to investigate, for the first time, the antiobesity activity of CG and its potential molecular mechanism in 3T3-L1 cells. METHODS: To study the effect of CG on adipogenesis, differentiating 3T3-L1 cells were treated every day with CG at various concentrations (0-100 µg/ml) for six days. Oil-red O staining and triglyceride content assay were performed to determine the lipid accumulation in 3T3-L1 cells. The expression of mRNAs or proteins associated with adipogenesis was measured using RT-PCR and Western blotting analysis. We examined the effect of CG on level of phosphorylated Akt in 3T3-L1 cells treated with CG at various concentration s during adipocyte differentiation. RESULTS: Differentiation was investigated with an Oil-red O staining assay using CG-treated 3T3-L1 adipocytes. We found that CG suppressed lipid droplet formation and adipocyte differentiation in 3T3-L1 cells in a dose-dependent manner. Treatment of the 3T3-L1 adipocytes with CG resulted in an attenuation of the expression of adipogenesis-related factors and lipid metabolic genes. The expression of C/EBPα and PPARγ, the central transcriptional regulators of adipogenesis, was decreased by the treatment with CG. The expression of genes involved in lipid metabolism, aP2 were significantly inhibited following the CG treatment. Moreover, the CG treatment down-regulated the phosphorylation levels of Akt and GSK3ß. CONCLUSIONS: Taken collectively, these data indicated that CG exerts antiadipogenic activity by inhibiting the expression of C/EBPß, C/EBPα, and PPARγ and the Akt signaling pathway in 3T3-L1 adipocytes.

Citrus aurantium flavonoids inhibit adipogenesis through the Akt signaling pathway in 3T3-L1 cells.

BACKGROUND: Obesity is a health hazard that is associated with a number of diseases and metabolic abnormalities, such as type-2 diabetes, hypertension, dyslipidemia, and coronary heart disease. In the current study, we investigated the effects of Citrus aurantium flavonoids (CAF) on the inhibition of adipogenesis and adipocyte differentiation in 3T3-L1 cells. METHODS: During adipocyte differentiation, 3T3-L1 cells were treated with 0, 10, and 50 µg/ml CAF, and then the mRNA and protein expression of adipogenesis-related genes was assayed. We examined the effect of CAF on level of phosphorylated Akt in 3T3-L1 cells treated with CAF at various concentrations during adipocyte differentiation. RESULTS: The insulin-induced expression of C/EBPß and PPARγ mRNA and protein were significantly down-regulated in a dose-dependent manner following CAF treatment. CAF also dramatically decreased the expression of C/EBPα, which is essential for the acquisition of insulin sensitivity by adipocytes. Moreover, the expression of the aP2 and FAS genes, which are involved in lipid metabolism, decreased dramatically upon treatment with CAF. Interestingly, CAF diminished the insulin-stimulated serine phosphorylation of Akt (Ser473) and GSK3ß (Ser9), which may reduce glucose uptake in response to insulin and lipid accumulation. Furthermore, CAF not only inhibited triglyceride accumulation during adipogenesis but also contributed to the lipolysis of adipocytes. CONCLUSIONS: In the present study, we demonstrate that CAF suppressed adipogenesis in 3T3-L1 adipocytes. Our results indicated that CAF down-regulates the expression of C/EBPß and subsequently inhibits the activation of PPARγ and C/EBPα. The anti-adipogenic activity of CAF was mediated by the inhibition of Akt activation and GSK3ß phosphorylation, which induced the down-regulation of lipid accumulation and lipid metabolizing genes, ultimately inhibiting adipocyte differentiation.

Identification of trophoblast-specific binding sites for GATA-2 that are essential for rat placental lactogen-I gene expression.

We identified a 3.4-kb 5'-flanking region of the rPL-I gene and examined its promoter activity using rat trophoblast Rcho-1 cells. A regulatory element between base pairs (bp) -2,487 and -2,310 in the 5'-flanking region was essential for maximum promoter activity of the rPL-I gene. This regulatory element was further characterized between bp -2,443 to -2,415 and -2,374 to -2,345. Electrophoretic mobility shift analysis showed that the interaction of nuclear extract proteins from differentiated Rcho-1 cells was inhibited by competition with a GATA-like sequence in the promoter, but not by a mutated GATA sequence. Moreover, the promoter activity of 2487 eLuc containing two novel GATA sites was significantly elevated by co-transfection of a GATA-2 expression vector in proliferating Rcho-1 cells. Our results demonstrate that GATA-2 is involved in multiple promoter regions to activate the specific expression of the rPL-I gene in placental tissue.

Avirulence gene diversity of Xanthomonas axonopodis pv. glycines isolated in Korea.

The hybridization patterns with the avrBs3 gene that is known to determine the recognition of host specificity were used to study the diversity of Xanthomonas axonopodis pv. glycines causing bacterial leaf pustule in soybean. A total of 155 strains were isolated from diverse tissues of soybean cultivars collected in Korea and were classified into six different type strains of OcsF, SL1017, SL1018, SL1045, SL1157, and SL2098 according to the patterns of avrBs3-homologous bands. When these type strains were inoculated on various cultivars, most of the Korean strains mildly induced disease symptoms on the resistant CNS1 cultivars. Unlike other type strains, strain SL2098, which appeared not to contain any avrBs3 homolog, induced only a few pustules on even highly susceptible cultivars. When a plasmid carrying the 3.7-kb avrBs3-homologous gene from strain SL1045 was introduced into SL2098, the transformant could not recover the pathogenicity in susceptible host plants. However, when avrBs3-homologous genes of strain SL1018 were mutated by transposon mutagenesis, one of the mutants in which a 5.2-kb chromosomal band homologous to avrBs3 was disrupted could not induce the hypersensitive response on resistant cultivars such as William82 or CNS2. Our results suggest that the avrBs3 homologs may play important roles in the pathogenicity of Xanthomonas axonopodis pv. glycines and the recognition of soybean cultivars.

Isolation and structural identification of a novel minoxidil analogue in an illegal dietary supplement: triaminodil.

A new minoxidil analogue was detected in an illegal dietary supplement advertised as a hair-growth treatment. The analogue was identified using ultra-performance liquid chromatography (UPLC), high-resolution mass spectrometry (LC-HR-MS) and nuclear magnetic resonance (NMR) spectroscopy. The compound was structurally elucidated as a minoxidil analogue in which the piperidinyl group of minoxidil was replaced with a pyrrolidinyl group corresponding to a molecular formula of C8H13N5O. The new analogue has been named triaminodil. As this is the first report of the compound, there are no chemical, toxicology or pharmacological data available.

Determination of Miroestrol and Isomiroestrol From Pueraria mirifica (White Kwao Krua) in Dietary Supplements by LC-MS-MS and LC-Q-Orbitrap/MS.

The purpose of this study is to develop LC-MS-MS and LC-Q-Orbitrap/MS method for the analysis of the components of Pueraria mirifica, which are illegal additives in dietary supplements. Blank samples and samples spiked with miroestrol and isomiroestrol were used for the initial development and validation studies. Specificity, linearity, limit of quantification (LOQ), limit of detection (LOD), accuracy, precision, recovery and stability were employed as the validation parameters. The LODs of miroestrol and isomiroestrol were found to be 4.17 and 0.84 ng/mL, respectively, whereas their LOQs were 12.50 and 2.52 ng/mL, respectively. The determination coefficient was over 0.999, intra- and inter-day precisions were 0.8-6.9 and 1.9-9.8%, respectively, and intra and inter-day accuracies were 82.1-103.7 and 85.0-109.7%, respectively. The mean recoveries of the targeted compounds from the dietary supplements ranged from 86.9 to 108.9%. The relative standard deviations (RSDs) for recovery were <5.8%. On the other hand, the RSD of stability was <11.0%. Eight dietary supplements were tested using the newly developed and validated method, out of which six were found to be adulterated samples.

A rapid method for the simultaneous determination of 25 anti-hypertensive compounds in dietary supplements using ultra-high-pressure liquid chromatography.

A novel, stable, simple and specific ultra-performance liquid chromatography method with ultraviolet detection (205 nm) for the simultaneous analysis of 25 anti-hypertensive substances was developed. The method was validated according to the International Conference of Harmonisation guidelines with respect to linearity, accuracy, precision, limit of detection (LOD), limit of quantitation (LOQ) and stability. From the ultra-performance liquid chromatography results, we identified the LOD and LOQ of solid samples to be 0.20-1.00 and 0.60-3.00 µg ml-1, respectively, while those of liquid samples were 0.30-1.20 and 0.90-3.60 µg ml-1, respectively. The linearity exceeded 0.9999, and the intra- and inter-day precisions were 0.15-6.48% and 0.28-8.67%, respectively. The intra- and inter-day accuracies were 82.25-111.42% and 80.70-115.64%, respectively, and the stability was lower than 12.9% (relative standard deviation). This method was applied to the monitoring of 97 commercially available dietary supplements obtained in Korea, such as pills, soft capsules, hard capsules, liquids, powders and tablets. The proposed method is accurate, precise and of high quality, and can be used for the routine, reproducible analysis and control of 25 anti-hypertensive substances in various dietary supplements. The work presented herein may help to prevent incidents related to food adulteration and restrict the illegal food market.

Screening for Corticosteroid Adulterants in Korean Herbal Medicines.

The objective of this study was to determine the presence of corticosteroids in illegal herbal medicines using ultra-high-performance liquid chromatography-tandem mass spectrometry. We collected 212 herbal medicine samples that were advertised as being effective for treatment of joint pain and bone aches. Samples were from the Korean commercial market during a span of four years (2010-2013), and the method was validated. The limits of quantification ranged from 0.47 to 15.0 ng/mL, and recoveries ranged from 80.6% to 119.5%. The intra- and interday precision ranged from 0.18% to 8.82% and from 0.09% to 8.96%, respectively. Among the samples, three samples (1.4%) were identified as adulterants. Dexamethasone was the only compound detected in the adulterated products. As the corticosteroid-adulteration of herbal medicines may become a major problem and lead to side effects, the continued development of screening procedures for herbal medicines is critical.

Development and validation of UPLC and LC-MS/MS methods for the simultaneous determination of anti-obesity drugs in foods and dietary supplements.

Recently, the number of the cases in which weight loss products have been sold with illegal adulterants has increased, as awareness of the problems of obesity grows. In this study, we developed simultaneous analysis methods to rapidly and accurately identify ingredients illegally mixed with foods and dietary supplements. Twenty-three anti-obesity drugs in foods and dietary supplements were determined by developed and validated UPLC and LC-MS/MS methods. The UPLC method were validated for the LOD and LOQ in the ranges 0.05-3.0 and 0.2-10.0 µg/mL, respectively. The determination coefficient was over 0.999, precision was <6.2 %, and the accuracy was 80.8-103.9 %. The mean recoveries ranged from 80.3 to 109.3 % and RSD of stability was less than 2.1 %. The determination of the 23 anti-obesity drugs was accomplished by electrospray ionization LC-MS/MS using multiple reaction monitoring (MRM). The LODs and LOQs were in the ranges 0.03-7.5 and 0.09-30.0 ng/mL, respectively. The LC-MS/MS method was validated for linearity (r(2) > 0.99), precision (RSD < 10.7 %), accuracy (94.1-109.1 %), recovery (80.5-113.5 %), and the RSD of stability was <7.8 %. Using the newly developed and validated method, 193 samples were tested, and 55 were found to be adulterated.

Simultaneous Analysis of Cannabinoid and Synthetic Cannabinoids in Dietary Supplements Using UPLC with UV and UPLC-MS-MS.

The primary purpose of this study was to develop and validate a method based on UPLC with UV and UPLC-MS-MS for the simultaneous analysis of different cannabinoids and synthetic cannabinoids in food as well as in herbal and dietary supplements. The limits of detection and quantitation of the method ranged from 0.1 to 0.3 and 0.3 to 0.9 µg/mL by UPLC with UV, respectively. The coefficient of determination was >0.999; the intra- and interday precision of the method were 0.1-3.7 and 0.9-4.1%, respectively. The intra- and interday accuracy were 94.8-103.1 and 98.3-100.9%, respectively. The mean recoveries of nine cannabinoids obtained from tablet samples ranged from 81.1 to 105.4%. The mean extraction recoveries of nine target cannabinoids obtained from various types of samples (tablets, capsules, powders, liquids, cookies and candies) ranged from 82.26 to 112.40%. The relative standard deviation (RSD) of the stability of the prepared sample solutions was <1.80%. Identification and quantification of the nine cannabinoids were accomplished by ion spray UPLC-MS-MS using multiple reaction monitoring. The UPLC-MS-MS method was validated for linearity (R(2) > 0.99); the precision was 0.1-4.0% (intraday) and 0.1-2.8% (interday), and the accuracy was 98.0-103.5% (intraday) and 97.1-103.2% (interday). The mean extraction recoveries of six types of samples were 82.2-114.5% and the RSD of stability was <6.54%, complying with the established international guidelines. The results indicated that the method can be used for rapid and accurate screening of cannabinoids present in food.

LC-ESI-MS/MS analysis of phosphodiesterase-5 inhibitors and their analogues in foods and dietary supplements in Korea.

A number of 188 food and dietary supplement samples were collected from 2009 to the first half of 2013 in Korean online and offline stores. A method to identify phosphodiesterase-5 (PDE-5) inhibitors and their analogues using liquid chromatography-electrospray ionisation-mass spectrometry/mass spectrometry (LC-ESI-MS/MS) was validated. Limit of detection and limit of quantitation of liquid-type and solid-type negative samples ranged from 0.05 to 3.33 ng/mL or ng/g and from 0.15 to 10.00 ng/mL or ng/g, respectively. Recoveries ranged from 83% to 112%. Nineteen PDE-5 inhibitors and their analogues were detected, with tadalafil group compounds being the most frequently observed (53.0%), followed by the sildenafil group (42.5%). Tadalafil concentrations ranged from 0.08 to 138.69 mg/g. Compounds were most frequently detected in capsules (in 40 of 80 adulterated samples). To protect public health and food safety, appropriate monitoring of PDE-5 inhibitors and their analogues in foods and dietary supplements is recommended.

Determination of anabolic-androgenic steroid adulterants in counterfeit drugs by UHPLC-MS/MS.

Anabolic-androgenic steroids (AASs) have been illegally used in counterfeit drugs to improve the performance of athletes. In addition, AASs have been used for cosmetic purpose by non-athletes. To determine the presence of 26 AASs, an analysis method using ultra-liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was developed and validated. The validated method was applied to 19 counterfeit drugs collected from the Internet and off-line markets during 2014. Nearly 50% (9/19) of the samples contained one of these 26 AASs. In addition, the concentration ranges of the AASs ranged from 0.09 to 119,228.57 mg/kg in the suspected samples. The determined AASs primarily consisted of testosterone and testosterone 17-propionate (26%) followed by boldenone (21%). These results indicate the adulteration of over-the-counter counterfeit drugs, and the continuous monitoring of counterfeit drugs or dubious dietary supplements containing anabolic steroids is warranted.

Identification of a new tadalafil analogue in an adulterated dietary supplement: trans-Bisprehomotadalafil.

A new tadalafil analogue was identified along with homotadalafil during routine screening of an adulterated dietary supplement using HPLC-DAD. The UV spectrum of this analogue was almost identical with that of tadalafil. This compound was isolated from the supplement by using semi-preparative HPLC and its structure was subsequently elucidated by performing Q-TOF/MS/MS and NMR spectroscopic experiments. The spectral data indicate that this tadalafil analogue is a dimeric compound that consists of an ethylamino group and two pretadalafil moieties. NOE experiments and comparison with (1)H NMR spectra of tadalafil and trans-tadalafil suggested the trans-relationship between the substituents on piperidine rings in the pretadalafil moieties.

Isolation and identification of novel propoxyphenyl thiosildenafil found in natural health food product.

A novel phosphodiesterase-5 (PDE-5) inhibitor was found in a natural health food product. The previously unknown sildenafil analogue was isolated using preparative HPLC. The structure of the compound was elucidated using HPLC with diode array detection (DAD), time-of-flight mass spectrometry (TOF/MS), and nuclear magnetic resonance spectroscopy (NMR). An [M + H](+) ion was detected at m/z 505.2077 by LC-TOF/MS that was consistent with C23H32N6O3S2 (-0.98 ppm). By NMR analysis, the analogue was identified as 1-methyl-5-(5-(4-methylpiperazin-1-ylsulfonyl)-2-propoxyphenyl)-3-propyl-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-thione. In this structure, the ethoxy group of thiosildenafil was substituted by a propoxy group of the unknown compound. Therefore, this novel thiosildenafil analogue was named propoxyphenyl thiosildenafil.

Determination of non-opioid analgesics in adulterated food and dietary supplements by LC-MS/MS.

Commercially available non-opioid analgesics such as acetaminophen and non-steroidal anti-inflammatory drugs (NSAIDs) have been used to adulterate some foods and dietary supplements. Considering the rapid growth of the dietary supplement market, it is essential to analyse various analgesics used for adulteration over a time period. Acetaminophen and 16 NSAIDs used to adulterate food and dietary supplements were simultaneously determined by LC-MS/MS. The method was validated by determining the coefficient of determinations, limit of quantification and recovery, and samples were analysed for the determination of analgesics. Consequently, acetaminophen, diclofenac, ibuprofen, indomethacin, naproxen and piroxicam were detected in 53 samples (n = 214). Ibuprofen was the most commonly used adulterant, which was detected in a wide concentration range (1.06-233.40 mg g(-1)) and was present in about one-third of the adulterated samples. Various types of samples, in particular pills and capsules (73.6% of the total positive samples), were found to be adulterated with non-opioid analgesics. Samples containing high concentrations of analgesics can have a deleterious effect on human health, and thus the continued monitoring of adulterated food and dietary supplements is essential to maintain a healthy life.

Monitoring of 29 weight loss compounds in foods and dietary supplements by LC-MS/MS.

Because of the rapid growth in dietary supplement availability and public concern for weight control, the investigation of foods and various dietary supplements illegally adulterated with weight loss compounds has become increasingly important. A total of 29 weight loss compounds, including sennoside, sibutramine, ephedrine and their analogues, found to be adulterated in foods and dietary supplements were simultaneously examined by LC-MS/MS. The 188 samples were collected between 2009 and 2012 in South Korea, and method validation was performed to determine the adulterants to the weight loss compounds. LODs, LOQs and linearity ranged from 0.03 to 7.5 ng ml⁻¹, from 0.08 to 30.00 ng ml⁻¹, and from 0.990 to 0.999, respectively. The results showed that nine weight loss compounds, namely bisacodyl, desmethylsibutramine, didesmethylsibutramine, ephedrine, fluoxetine, pseudoephedrine, sennoside A, sennoside B and sibutramine, were detected in 62 of all collected samples and were found in order of frequency as follows: sibutramine, 25.7%; sennoside A, 22.9%; sennoside B, 20.0%; fluoxetine, 8.6%; desmethylsibutramine, 7.1%; bisacodyl, ephedrine, and pseudoephedrine, 4.3%; and didesmethylsibutramine, 2.9%. Sibutramine, which was the most frequently found adulterant, ranged in levels from 0.03 to 132.40 mg g⁻¹ (2010), from 0.88 to 76.2 mg g⁻¹ (2011), and from 0.07 to 0.24 mg g⁻¹ (2012). Although the concentrations of most compounds ranged widely, some compounds such as bisacodyl and fluoxetine were found at high concentrations in several samples.