The Development of One New Normal Phase Liquid Chromatography Method and Thermodynamic Investigation of Olodaterol Hydrochloride Enantiomer.

In order to improve and replace the enantiomer method outlined in the olodaterol hydrochloride draft monograph (From the European Pharmacopoeia forum), one new, simple, and fast enantioselective normal phase high-performance liquid chromatography chiral method was developed on polysaccharide-based Chiral MX (2) (4.6 × 250 mm, 5 µm) column. n-Hexane, ethanol, and diethylamine in the ratio of 40:60:0.1 (V/V/V) were selected as mobile phase at a flow rate of 0.8 mL/min, and the detection was performed on a photodiode array detector at 225 nm with 5 µL injection volume. The column temperature was set at 40°C for better peak shape and sensitivity. The analysis time can be shortened to 15 min, whereas the resolution between enantiomer and olodaterol was found to be even more than 10.0, which was far better than that obtained with the reported method in this draft monograph. The developed chiral method was validated in accordance with ICH Q2 (R1), including specificity, LOD&LOQ, precision, linearity, accuracy, and robustness. Thereby, the proposed method was demonstrated to be suitable for the determination of enantiomer in olodaterol hydrochloride bulk drug and drug product. Besides, the thermodynamic parameters were evaluated on the basis of Van't Hoff plots that was used to explain correlative chiral recognition mechanisms with the chiral stationary phase.

Optimizing removal of antiretroviral drugs from tertiary wastewater using chlorination and AI-based prediction with response surface methodology.

Chemical and pharmaceutical chemicals found in water sources create substantial risks to human health and the environment. The presence of pharmaceutical contaminants in water can cause antibiotic resistance development, toxicity to aquatic organisms, and endocrine disruption. Hence, the elimination of chemicals and other contaminants from wastewater prior to its release is a burgeoning concern in the domains of engineering and science. The use of treatment technologies in wastewater treatment plants can remove pharmaceutical contaminants through the oxidation process. However, many traditional wastewater treatment plants lack the advanced monitoring tools required to detect low concentrations of pharmaceuticals. Without the ability to detect these compounds, it's challenging to treat them effectively. The goal of this study was to use Response Surface Methodology (RSM) and Artificial Neural Networks (ANN) algorithms to model and improve how Nevirapine and Efavirenz break down in different chlorination conditions. The RSM analysis revealed statistically significant models (F-values: Nevirapine, pH-t: 108.15, T-t: 76.55, ICC-t: 110.84), indicating a strong correlation between operational parameters (pH, temperature, and initial chlorine concentration) and degradation behavior. The ANN model accurately predicted the degradation of both Nevirapine and Efavirenz under various chlorination conditions, as confirmed by analyzing actual-predicted graphs, residual plots, and Mean Squared Error (MSE) values. The ANN model using ICC-t achieved the highest MOD value of 31.31 % for Nevirapine. The ANN model based on ICC-t yielded a maximum MOD value of 16.06 % for Efavirenz. These findings provide valuable insights into optimizing chlorination processes for better removal of these pharmaceutical contaminants from water.

Oxindole and Benzoxazinone Alkaloids from the Seeds of Persea americana (Avocado) and Their SIRT1 Stimulatory Activity.

Persea americana Mill. (Lauraceae), commonly known as avocado, is a well-known food because of its nutrition and health benefits. The seeds of avocado are major byproducts, and thus their phytochemicals and bioactivities have been of interest for study. The chemical components of avocado seeds were investigated by using UPLC-qTOF-MS/MS-based molecular networking, resulting in the isolation of seven new oxindole alkaloids (1-7) and two new benzoxazinone alkaloids (8 and 9). The chemical structures of the isolated compounds were identified by the analysis of NMR data in combination with computational approaches, including NMR and ECD calculations. Bioactivities of the isolated compounds toward silent information regulation 2 homologue-1 (SIRT1) in HEK293 cells were assessed. The results showed that compound 1 had the most potent effect on SIRT1 activation with an elevated NAD+/NADH ratio with potential for further investigation as an anti-aging agent.

Quantification of nine antiretroviral drugs in cerebrospinal fluid: An approach to overcome sample collection tube adsorption.

A highly sensitive LC-MS/MS methods were developed and validated to quantify nine antiretrovirals (atazanavir [ATV], tenofovir [TFV], emtricitabine [FTC], darunavir [DRV], dolutegravir [DTG], efavirenz [EFV], lamivudine [3TC], raltegravir [RAL], and ritonavir [RTV]) in human cerebral spinal fluid (CSF). The approach remedies adsorption issues caused by polypropylene based sample collection tubes. 1% ammonium hydroxide in methanol was added in an amount equal to the volume of each quality control (QC) or patient sample. Protein precipitation was utilized with a CSF sample volume of 100 µL and a 100 µL of methanol:ACN and vortexed. Chromatographic separation was achieved with a 3 × 100 ACE® C18 column for ATV, DRV, DTG, EFV, RTV and RAL, and a 2 × 100 Polar RP column for TFV/FTC/3TC. Mobile phase was methanol:water:formic acid (70:30:0.1, v/v/v) for ATV, DRV, DTG, EFV and RTV (10 uL injection, flow rate: 1.00 mL/min), ACN:water:formic acid (35:65:0.1, v/v/v) for RAL (50 uL injection, flow rate: 1.00 mL/min), ACN:water:formic acid (2:98:0.1, v/v/v) for TFV, FTC and 3TC (50 uL injection, flow rate: 0.35 mL/min). Column temperature was 40° C across all assays. The mass spectrometer was operated in positive, multiple-reaction-monitoring (MRM) mode with electrospray ionization (ESI) for all analytes with the exception of EFV, which was operated in negative, MRM mode with ESI. The assay was linear over the calibration range of 1 to 250 ng/mL for all analytes. The addition of 1% ammonium hydroxide in sample tubes overcame up to 44% negative bias in QC samples and allowed the methods to meet full validation criteria.

Exploration of multifunctional wood coating based on an interpenetrating network system of rosin-CO2-based polyurethane and mussel bionic rosin-based benzoxazine.

Polyurethane (PU) prepared by blending rosin base and CO2-polyol already has good mechanical properties and hydrophobic effect and has powerful benefits in acid and alkali resistance and salt resistance. In this study, mussel bionic rosin-based benzoxazine (BZ) was synthesized using dehydroabietylamine, catechol, and paraformaldehyde. Mixing BZ into PU can endow the resulting PU/BZ with special effects such as zero curing shrinkage, excellent mechanical behavior, and flame retardancy through a 3D interpenetrating network system. From the results, the modulus of rupture (MOR) and modulus of elasticity (MOE) of PU wood coatings are 97.04 and 2601.97 MPa, respectively; in contrast, the PU/BZ wood coatings exhibited higher values of MOR and MOE of 110.87 and 2738.11 MPa. PU/BZ wood coatings show higher flexural strength and elastic modulus. They are also stronger than PU coatings in terms of acid/alkali and aging resistance. At the same time, the coating is endowed with flame retardant properties, and the LOI is 30.2 due to the presence of BZ. Thus, PU/BZ can be a versatile and practical wood coating. The interpenetrating network system of PU/BZ has an innovative impact on the preparation of wood coatings.

3D Printed Microfluidic Device for Magnetic Trapping and SERS Quantitative Evaluation of Environmental and Biomedical Analytes.

Surface-enhanced Raman scattering (SERS) is an ideal technique for environmental and biomedical sensor devices due to not only the highly informative vibrational features but also to its ultrasensitive nature and possibilities toward quantitative assays. Moreover, in these areas, SERS is especially useful as water hinders most of the spectroscopic techniques such as those based on IR absorption. Despite its promising possibilities, most SERS substrates and technological frameworks for SERS detection are still restricted to research laboratories, mainly due to a lack of robust technologies and standardized protocols. We present herein the implementation of Janus magnetic/plasmonic Fe3O4/Au nanostars (JMNSs) as SERS colloidal substrates for the quantitative determination of several analytes. This multifunctional substrate enables the application of an external magnetic field for JMNSs retention at a specific position within a microfluidic channel, leading to additional amplification of the SERS signals. A microfluidic device was devised and 3D printed as a demonstration of cheap and fast production, with the potential for large-scale implementation. As low as 100 µL of sample was sufficient to obtain results in 30 min, and the chip could be reused for several cycles. To show the potential and versatility of the sensing system, JMNSs were exploited with the microfluidic device for the detection of several relevant analytes showing increasing analytical difficulty, including the comparative detection of p-mercaptobenzoic acid and crystal violet and the quantitative detection of the herbicide flumioxazin and the anticancer drug erlotinib in plasma, where calibration curves within diagnostic concentration intervals were obtained.

Comprehensive profiling of semi-polar phytochemicals in whole wheat grains (Triticum aestivum) using liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry.

INTRODUCTION: Wheat (Triticum aestivum) it is one of the most important staple food crops worldwide and represents an important resource for human nutrition. Besides starch, proteins and micronutrients wheat grains accumulate a highly diverse set of phytochemicals. OBJECTIVES: This work aimed at the development and validation of an analytical workflow for comprehensive profiling of semi-polar phytochemicals in whole wheat grains. METHOD: Reversed-phase ultra-high performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (UHPLC/ESI-QTOFMS) was used as analytical platform. For annotation of metabolites accurate mass collision-induced dissociation mass spectra were acquired and interpreted in conjunction with literature data, database queries and analyses of reference compounds. RESULTS: Based on reversed-phase UHPLC/ESI-QTOFMS an analytical workflow for comprehensive profiling of semi-polar phytochemicals in whole wheat grains was developed. For method development the extraction procedure and the chromatographic separation were optimized. Using whole grains of eight wheat cultivars a total of 248 metabolites were annotated and characterized by chromatographic and tandem mass spectral data. Annotated metabolites comprise hydroquinones, hydroxycinnamic acid amides, flavonoids, benzoxazinoids, lignans and other phenolics as well as numerous primary metabolites such as nucleosides, amino acids and derivatives, organic acids, saccharides and B vitamin derivatives. For method validation, recovery rates and matrix effects were determined for ten exogenous model compounds. Repeatability and linearity were assessed for 39 representative endogenous metabolites. In addition, the accuracy of relative quantification was evaluated for six exogenous model compounds. CONCLUSIONS: In conjunction with non-targeted and targeted data analysis strategies the developed analytical workflow was successfully applied to discern differences in the profiles of semi-polar phytochemicals accumulating in whole grains of eight wheat cultivars.

Root-Exuded Benzoxazinoids: Uptake and Translocation in Neighboring Plants.

Plants have evolved advanced chemical defense mechanisms, including root exudation, which enable them to respond to changes occurring in their surroundings rapidly. Yet, it remains unresolved how root exudation affects belowground plant-plant interactions. The objective of this study was to elucidate the fate of benzoxazinoids (BXs) exuded from the roots of rye (Secale cereale L.) plants grown with hairy vetch (Vicia villosa). A rapid method that allows nondestructive and reproducible chemical profiling of the root exudates was developed. Targeted chemical analysis with high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was performed to investigate the changes in the composition and concentration of BXs in the rye plant, and its root exudate in response to cocultivation with hairy vetch. Furthermore, hairy vetch plants were screened for the possible uptake of BXs from the rhizosphere and their translocation to the shoot. Rye significantly increased the production and root exudation of BXs, in particular 2-ß-d-glucopyranosyloxy-4-hydroxy-1,4-benzoxazin-3-one (DIBOA-glc) and 2-ß-d-glucopyranosyloxy-4-hydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA-glc), in response to cocultivation with hairy vetch. DIBOA-glc and DIMBOA-glc were absorbed by the roots of the cocultivated hairy vetch plants and translocated to the shoots. These findings will strongly improve our understanding of the exudation of BXs from the rye plant and their role in interaction with other plant species.

Determination of selected antiretroviral drugs in wastewater, surface water and aquatic plants using hollow fibre liquid phase microextraction and liquid chromatography - tandem mass spectrometry.

This work describes a simple and sensitive method for the simultaneous isolation, enrichment, identification and quantitation of selected antiretroviral drugs; emtricitabine, tenofovir disoproxil and efavirenz in aqueous samples and plants. The analytical method was based on microwave extraction and hollow fibre liquid phase microextraction technique coupled with ultra-high pressure liquid chromatography-high resolution mass spectrometry. A multivariate approach via a half-fractional factorial design was used focusing on six factors; donor phase pH, acceptor phase HCl concentration, extraction time, stirring rate, supported liquid membrane carrier composition and salt content. The optimal enrichment factors for emtricitabine, tenofovir disoproxil and efavirenz from aqueous phase were 78, 111 and 24, respectively. The analytical method yielded recoveries in the range of 86 to 111%, and quantitation limits for emtricitabine, tenofovir disoproxil and efavirenz in wastewater were 0.033, 0.10 and 0.53 µg L-1, respectively. The drugs were detected in most samples with concentrations up to 37.6 µg L-1 recorded for efavirenz in wastewater effluent. Roots of the water hyacinth plant had higher concentrations of the investigated drugs ranging from 7.4 to 29.6 µg kg-1. Overall, hollow fibre liquid phase microextraction proved to be an ideal tool for isolating and pre-concentrating the selected antiretroviral drugs from environmental samples.

Synthesis and application of a molecularly imprinted polymer in selective solid-phase extraction of efavirenz from water.

Efavirenz is one of the antiretroviral drugs widely used to treat the human immunodeficiency virus. Antiretroviral drugs have been found to be present in surface water and wastewater. Due to complexity of environmental samples, solid-phase extraction (SPE) is used for isolation and pre-concentration of antiretroviral drugs prior to their chromatographic analysis. However, the commercially available SPE sorbents lack selectivity, which tends to prolong the analysis time. Therefore, in this study a molecularly imprinted polymer was synthesized for the specific recognition of efavirenz and then applied as the SPE sorbent for its extraction from wastewater and surface water samples. The imprinted and non-imprinted polymers were synthesized using a bulk polymerization technique where efavirenz was used as the template, 2-vinylpyridine as functional monomer, 1,1'-azobis-(cyclohexanecarbonitrile) as initiator, ethylene glycol dimethacrylate as cross-linker and toluene:acetonitrile (9:1, v/v) as the porogenic solvent mixture. The characterization was performed using Fourier transform infrared spectroscopy, scanning electron microscopy, Brunauer-Emmett-Teller, elemental analysis, and thermogravimetric analysis techniques. Results showed better selectivity of molecularly imprinted polymer to efavirenz than did non-imprinted polymer. The analysis was performed using high performance liquid chromatography equipped with a photo-diode array detector. The analytical method gave a detection limit of 0.41 µg/L and the analyte recovery of 81% in wastewater. The concentrations found in wastewater ranged from 2.79 to 120.7 µg/L, while in surface water they were between 0.975 and 2.88 µg/L. Therefore, the results of this study show a strong need for a detailed screening of efavirenz in major water utilities in the country.

Solid-phase extraction combined with dispersive liquid-liquid microextraction/HPLC-UV as a sensitive and efficient method for extraction, pre-concentration and simultaneous determination of antiretroviral drugs nevirapine, efavirenz and nelfinavir in pharmaceutical formulations and biological samples.

Therapeutic drug monitoring (TDM) of antiretroviral drugs requires accurate and precise analysis of their trace amounts in plasma samples. Solid-phase extraction (SPE) coupled with dispersive liquid-liquid microextraction based on solidification of floating organic drop (DLLME-SFO) was introduced as a simple and sensitive method for extraction, pre-concentration and simultaneous determination of efavirenz (EFV), nelfinavir (NFV) and nevirapine (NVP) in human plasma and pharmaceutical formulations by using high performance liquid chromatography (HPLC) with UV detection. A response surface methodology (RSM) based on central composite design (CCD) was used for optimizing the main variables in the extraction procedure. Under the optimum experimental conditions, the calibration curves were linear in the range of 0.1-400 ng mL-1 with correlation coefficients from 0.9982 to 0.9997 and limits of detection (at S/N = 3) of 0.03 to 0.07 ng mL-1. Moreover the intra-day and inter-day precision (RSD%) were in the range of 2.2-4.2% and 3.1-5.2%, respectively. The enrichment factors and relative recoveries of the anti-HIV drugs were 459-1507 and 93.7-105.4%. The method was successfully applied to the simultaneous determination of the trace amounts of the antiretroviral drugs in blood plasma and pharmaceutical formulations.

Cereal aphids differently affect benzoxazinoid levels in durum wheat.

Aphids are major pests in cereal crops that cause direct and indirect damage leading to yield reduction. Despite the fact that wheat provides 20% of the world's caloric and protein diet, its metabolic responses to aphid attack, in general, and specifically its production of benzoxazinoid defense compounds are poorly understood. The objective of this study was to compare the metabolic diversity of durum wheat seedlings (Triticum turgidum ssp. durum) under attack by three different cereal aphids: i) the English grain aphid (Sitobion avenae Fabricius), ii) the bird cherry-oat aphid (Rhopalosiphum padi L.), and iii) the greenbug aphid (Schizaphis graminum Rondani), which are some of the most destructive aphid species to wheat. Insect progeny bioassays and metabolic analyses using chromatography/Q-Exactive/mass spectrometry non-targeted metabolomics and a targeted benzoxazinoid profile were performed on infested leaves. The insect bioassays revealed that the plants were susceptible to S. graminum, resistant to S. avenae, and mildly resistant to R. padi. The metabolic analyses of benzoxazinoids suggested that the predominant metabolites DIMBOA (2,4-dihydroxy-7-methoxy-1,4-benzoxazin- 3-one) and its glycosylated form DIMBOA-glucoside (Glc) were significantly induced upon both S. avenae, and R. padi aphid feeding. However, the levels of the benzoxazinoid metabolite HDMBOA-Glc (2-hydroxy-4,7-dimethoxy-1,4-benzoxazin-3-one glucoside) were enhanced due to the feeding of S. avenae and S. graminum aphids, to which Svevo was the most resistant and the most susceptible, respectively. The results showed a partial correlation between the induction of benzoxazinoids and aphid reproduction. Overall, our observations revealed diverse metabolic responses of wheat seedlings to cereal aphid feeding.

The use of experimental design for the development and validation of an HPLC-ECD method for the quantitation of efavirenz.

A high performance liquid chromatography with electrochemical detection (HPLC-ECD) method for the quantitation of efavirenz (EFV) was developed, since traditional HPLC-UV methods may be inappropriate, given that EFV undergoes photolytic degradation following exposure to UV light. This work describes the use of response surface methodology (RSM) based on a central composite design (CCD) to develop a stability-indicating HPLC method with pulsed ECD in direct current (DC) mode at an applied potential difference and current of +1400 mV and 1.0 µA for the analysis of EFV. Separation of EFV and imipramine was achieved using a Nova-Pak®C18 cartridge column and a mobile phase of phosphate buffer (pH 4.5): acetonitrile (ACN) (55:45 v/v). Mobile phase pH, buffer molarity, ACN concentration and applied potential difference were investigated. The optimized method produced sharp well resolved peaks for imipramine and EFV with retention times of 3.70 and 8.89 minutes. The calibration curve was linear (R2 = 0.9979) over the range 5-70 µg/mL. Repeatability and intermediate precision ranged between 3.37 and 4.34 % RSD and 1.31 and 4.29 % RSD and accuracy between -0.80 and 4.71 % bias. The LOQ and LOD were 5.0 and 1.5 µg/mL. The method was specific for EFV and was used to analyse EFV in commercially available tablets. The HPLC-ECD method is more suitable for quantitative analysis of EFV than HPLC-UV.

Simultaneous quantitation of zidovudine, efavirenz, lopinavir and ritonavir in human hair by liquid chromatography-atmospheric pressure chemical ionization-tandem mass spectrometry.

Nowadays, zidovudine, efavirenz, lopinavir and ritonavir are important components of the second-line antiretroviral therapeutic regimen of National Free Antiretroviral Treatment Program in China. The measurement of antiretroviral drugs in hair will facilitate for the evaluation of the long-term adherence to highly active antiretroviral therapy. Nevertheless, no study illustrated simultaneous quantitation of the four drugs in hair. The study intended to develop a simple, sensitive and selective method for simultaneous quantitation of the four drugs in hair. The detection employed high liquid chromatography tandem mass spectrometry with atmospheric pressure chemical ionization in positive mode. Hair samples were incubated in methanol at 40 °C for 16 h. The method showed the limit of detection at 18, 8, 5 and 6 pg/mg for zidovudine, efavirenz, lopinavir and ritonavir, respectively. The linear range (R2 > 0.99) was 36-5000 pg/mg for zidovudine, 16-5000 pg/mg for efavirenz, 10-50,000 pg/mg for lopinavir and 12-12,500 pg/mg for ritonavir. The intra-day and inter-day coefficients of variation were <15% and the recovery varied from 88.1 to 110.5%. The population analysis revealed that patients with high adherence showed significantly higher drug concentrations in hair than those with low adherence for both AZT and EFV (ps < 0.05). There was high association in drug contents in hair between AZT and EFV or LPV and RTV (ps < 0.05).

Bioactivity of Acanthus mollis - Contribution of benzoxazinoids and phenylpropanoids.

ETHNOPHARMACOLOGICAL RELEVANCE: Acanthus mollis is a plant native to the Mediterranean region, traditionally used as diuretic, anti-inflammatory and soothing of the mucous membranes of the digestive and urinary tract and externally as healing of wounds and burns, also demonstrating analgesic and anti-inflammatory activities. However, studies focused on its phytochemical composition as well as scientific proof of Acanthus mollis efficacy are scarce. AIM OF THE STUDY: The proposed work aims to perform a phytochemical characterization and evaluation of the therapeutic potential of Acanthus mollis, based on biological properties that support its traditional uses. MATERIAL AND METHODS: In this study, an 96% ethanol extract from Acanthus mollis leaves was obtained and its phytochemical composition evaluated using High Performance Liquid Chromatography with Photodiode Array Detector coupled to Electrospray Ionization Mass Spectrometry (HPLC-PDA-ESI/MSn). The chemical structure of the compound isolated was elucidated using 1H and 13C Nuclear Magnetic Resonance (NMR), 1H-correlation spectroscopy (1H-COSY), heteronuclear single quantum correlation (HSQC) and heteronuclear multiple-bond correlation (HMBC). The quantification of the constituents was performed using two external standards (2,4-dihydroxy-1,4-benzoxazin-3-one and verbascoside). The antioxidant activity was determined by the 2,2-diphenyl-1-pycrylhydrazyl (DPPH) assay. Anti-inflammatory activity was determined measuring the inhibition of nitric oxide production by RAW 264.7 macrophages stimulated with the TLR4 agonist lipopolysaccharide (LPS) and through lipoxygenase (LOX) inhibition assay. The cytotoxicity was screened on two lines (RAW 264.7 and HaCaT) using the resazurin assay. RESULTS: Compounds such as verbascoside and its derivatives, as well as benzoxazinoids were found as the main constituents. A percentage of 5.58% was verified for the 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA) derivatives. DIBOA was the main compound of the extract. Significant concentrations were also found for phenylpropanoids, which constitute about 4.39% of the total compounds identified. This extract showed antioxidant capacity against DPPH (IC50 = 40.00 ±â€¯1.59 µg/mL) and superoxide anion (IC50 = 29.42 ±â€¯1.99 µg/mL). It also evidenced anti-inflammatory potential in RAW 264.7 macrophages, presenting capacity for nitric oxide reduction (IC50 = 28.01 µg/mL). Moreover, in vitro studies have shown that this extract was able to inhibit the lipoxygenase, with an IC50 of 104.39 ±â€¯4.95 µg/mL. Importantly, all effective concentrations were devoid of cytotoxicity in keratinocytes, thus highlighting the safety of the extract for the treatment of skin inflammatory related diseases. Concerning macrophages it was also possible to disclose concentrations showing anti-inflammatory activity and without cytotoxicity (up to 30 µg/mL). The benzoxazinoid DIBOA demonstrated a considerable anti-inflammatory activity suggesting its important contribution to this activity. CONCLUSIONS: These results corroborate the anti-inflammatory properties traditionally attributed to this plant. Among the compounds identified in this study, benzoxazinoids exhibited a significant anti-inflammatory activity that was never previously described. Ethanol seems to be a good option for the extraction of these bioactive compounds, since relevant antioxidant/anti-radical and anti-inflammatory activities were found for this extract.

A validated liquid chromatography/tandem mass spectrometry method for the analysis of efavirenz in 0.2 mg hair samples from human immunodeficiency virus infected patients.

RATIONALE: Drug levels in hair provide a longer window of detection, compared to plasma drug levels, and therefore hair analysis has the advantage of assessing adherence over a longer period of time. No methods for the analysis of antiretroviral drugs in hair currently exist in South Africa, and worldwide there is only one validated method for the determination of efavirenz in hair that has been published. METHODS: Efavirenz was extracted from 0.2 mg of hair through a simultaneous pulverization and extraction step. Separation was achieved on an Agilent Poroshell C18 column using an isocratic elution with a total run time of 3 min. A triple quadrupole mass spectrometer set to electrospray ionization in positive multiple reaction monitoring mode was used for detection. The method was validated over the concentration range 0.625-40 ng/mg. RESULTS: Using ten times less hair than in a previously published method, the lower limit of quantitation was validated at 0.625 ng/mg. The interday and intraday assay precision, expressed as the percentage coefficient of variation (CV), for spiked calibration standards and quality control samples was lower than 7% and accuracy ranged from 97 to 110%. For quality controls prepared from authentic hair the CV was less than 12%. The extraction efficiency for authentic quality control samples was determined to be 83% after repeated extractions of the same samples. CONCLUSIONS: This paper reports the first quantitative method for the determination of efavirenz in hair to be developed in South Africa. The validated method allowed for the successful monitoring of efavirenz in hair collected from HIV-infected patients as part of a clinical study.

Quantification of benzoxazinoids and their metabolites in Nordic breads.

Benzoxazinoids (Bx) and their metabolites are molecules with suggested health effects in humans, found in cereal grains and consequently in cereal foods. However, to date little is known about the amount of Bx in our diet. In this study, deuterated standards 2-hydroxy-1,4-benzoxazin-3-one (HBOA-d4) and 2-hydroxy-N-(2-hydroxyphenyl) acetamide (HHPAA-d4) were synthesized, to allow quantification of nine Bx and their metabolites in 30 breads and flours from Nordic countries by UHPLC-MS/MS. Samples containing rye had larger amounts of Bx (143-3560µg/g DM) than the ones containing wheat (11-449µg/g DM). More Bx were found in whole grain wheat (57-449µg/g DM) compared to refined wheat (11-92µg/g DM) breads. Finnish sourdough rye breads were notably high in their 2-hydroxy-N-(2-hydroxyphenyl) acetamide (HHPAA) concentration (40-48µg/g DM). This new information on Bx content in flours and breads available in the Nordic countries will be useful for future work on determining dietary exposure to Bx.

Automated high throughput analysis of antiretroviral drugs in dried blood spots.

For therapeutic drug monitoring in remote settings, dried blood spots (DBS) are particularly advantageous, as blood sample collection and handling is uncomplicated. The aim of this study was to develop and validate an automated extraction method for the analysis of nevirapine, efavirenz and lopinavir in DBS samples. Automated extraction was performed with methanol : water (70 : 30 v/v), using a DBS-MS 500 autosampler coupled to a liquid chromatography tandem mass spectrometry system. The autosampler used digital images of each DBS to position the extraction head, sprayed 10 µl of internal standard onto each DBS and extracted a 4-mm disc (Ø) from the centre of each spot by unilateral flow using 25-µl extraction solvent. The analytes were baseline separated on a pentafluorophenyl column and analysed by using electrospray ionization with multiple reaction monitoring in positive polarity mode for nevirapine and lopinavir and in negative mode for efavirenz. The method was linear between 10 and 10 000 ng/ml for all analytes. Automated sample extraction resulted in consistent recoveries (nevirapine: 70 ± 6%, efavirenz: 63 ± 11% and lopinavir: 60 ± 10%) and matrix effects between different donors and concentration levels. Intra-day and inter-day accuracy and precision deviations were ≤15%. Manual and automated extractions of DBS samples collected within the framework of an adherence assessment study in rural Tanzania showed good agreements with deviations of less than 10%. Our study highlights that therapeutic drug monitoring samples obtained in the resource-constrained setting of rural Africa can be reliably determined by automated extraction of DBS. Overall, automatization improved method sensitivity and facilitates analysis of large sample numbers. Copyright © 2017 John Wiley & Sons, Ltd.

Determination of endocrine disrupting chemicals and antiretroviral compounds in surface water: A disposable sorptive sampler with comprehensive gas chromatography - Time-of-flight mass spectrometry and large volume injection with ultra-high performance liquid chromatography-tandem mass spectrometry.

Many rural dwellers and inhabitants of informal settlements in South Africa are without access to treated water and collect untreated water from rivers and dams for personal use. Endocrine disrupting chemicals (EDCs) have been detected in surface water and wildlife of South Africa. EDCs are often present in complex environmental matrices at ultra-trace levels complicating detection thereof. We report a simplified multi-residue approach for the detection and quantification of EDCs, emerging EDCs, and antiretroviral drugs in surface water. A low cost (less than one US dollar), disposable, sorptive extraction sampler was prepared in-house. The disposable samplers consisted of polydimethylsiloxane (PDMS) tubing fashioned into a loop which was then placed in water samples to concentrate EDCs and emerging pollutants. The PDMS samplers were thermally desorbed directly in the inlet of a GC, thereby eliminating the need for expensive consumable cryogenics. Comprehensive gas chromatography coupled to time-of-flight mass spectrometry (GC×GC-TOFMS) was used for compound separation and identification. Linear retention indices of EDCs and emerging pollutants were determined on a proprietary Crossbond® phase Rtx®-CLPesticides II GC capillary column. In addition, large volume injection of surface water into an ultra-performance liquid chromatograph tandem mass spectrometer (UPLC-MS/MS) was used as complementary methodology for the detection of less volatile compounds. Large volume injection reduced tedious and costly sample preparation steps. Limits of detection for the GC method ranged from 1 to 98pg/l and for the LC method from 2 to 135ng/l. Known and emerging EDCs such as pharmaceuticals, personal care products and pesticides, as well as the antiretroviral compounds, efavirenz and nevirapine, were detected in surface water from South Africa at concentration levels ranging from 0.16ng/l to 227ng/l.

Development and validation of a liquid chromatography-MS/MS method for simultaneous quantification of tenofovir and efavirenz in biological tissues and fluids.

Millions of people worldwide live with human immunodeficiency virus (HIV) infection thus justifying the continuous search for new prevention and treatment strategies, including topical microbicide products combining antiretroviral drugs (ARVs) such as tenofovir (TFV) and efavirenz (EFV). Therefore, the aim of this work was to develop and validate a high performance liquid chromatography method coupled to triple quadrupole-tandem mass spectrometry (HPLC-MS/MS) for the quantification of TFV and EFV in biological matrices (mouse vaginal tissue, vaginal lavage and blood plasma). Chromatographic separation was achieved using a reversed phase C18 column (3µm, 100×2.1mm) at 45°C and elution in gradient mode using a combination of 0.1% (v/v) formic acid in water and 0.1% (v/v) formic acid in acetonitrile at 0.35mLmin-1. Total run time was 9min, with retention time of 2.8 and 4.1min for TFV and EFV, respectively. The MS was operated in positive ionization mode (ESI+) for TFV and in negative ionization mode (ESI-) for EFV detection. Data were acquired in selected reaction monitoring (SRM) mode and deuterated ARVs were employed as internal standards. Calibration curves were linear for ARV concentrations ranging from 4 to 500ngmL-1 with LOD and LOQ for both analytes ≤0.4 and ≤0.7ngmL-1 in sample extracts, respectively. The method was found to be specific, accurate (96.0-106.0% of nominal values) and precise (RSD<2.4%) in all matrices. Both TFV and EFV were found to be stable in all matrices after standing 24h at room temperature (20°C) or in the autosampler, and after three freeze-thawing cycles. Mean recovery values of ARVs spiked in mice tissues or fluids were ≥88.4%. Matrix effects were observed for EFV determination in tissue and plasma extracts but compensated by the use of deuterated internal standards. The proposed methodology was successfully applied to a pharmacokinetic study following intravaginal administration of both ARVs.