Background: Efavirenz (EFV) is one of the preferred components of first line antiretroviral treatment. EFV is characterised by a long plasma half-life (40-55h) with large inter-patient variability which raises the potential for individualisation of therapy. Analyses of EFV levels in plasma require specialised facilities (cold storage/transport) which, in resource-limited settings (RLS), can be problematic; DBS-EFV measurements thus provide a cheap, easy alternative for therapeutic drug monitoring. Our aim was to develop and validate a LC-MS/MS method to quantify EFV in DBS collected as part of clinical trials in RLS.

Methods: DBS for standards, QC and patient samples, were excised and then extracted with ethyl acetate/n-hexane (50/50 v/v) after addition of internal standard (IS) hexobarbital, and 1mol/L K2CO3. The extract was evaporated to dryness, the residue reconstituted in mobile phase and analysed directly by LC-MS/MS. Gradient elution was on a reverse-phase C18 column using 1mmol/L ammonium acetate in water and acetonitrile. Quantification was by Selected Reaction Monitoring (SRM) in negative ionisation mode. DBS samples were obtained at several time-points over 24hr from HIV+ patients on either 400 or 600 mg EFV in combination with emtricitabine/tenofovir.

Results: The IS and EFV eluted at 2.68 and 3.54 minutes respectively in a 5 minute run time. Matrix effects were minimal (-5.4%). Calibration curves were validated over a concentration range of 25-5000 ng/mL. Intra and inter assay variation ranged between 6.7-8.7% for imprecision and 100.3-104.2% for accuracy. Mean recovery was >64%. The DBS data showed a strong positive correlation with a validated plasma EFV assay (R=0.9764, P<0.001). EFV concentrations from DBS were approximately 42% lower than the paired plasma values and the ratio of blood/plasma did not change over the dosing interval.

Conclusion: The validated assay is now routinely applied to clinical samples measuring DBS-EFV for PK analysis. The methodology is robust, accurate and sensitive.

BACKGROUND: Efavirenz (EFV) is one of the preferred components of first-line antiretroviral treatment. EFV is characterized by a long plasma half-life (40-55 hours) with large interpatient variability, which raises the potential for individualization of therapy. Analyses of EFV levels in plasma require specialized facilities (cold storage/transport) which, in resource-limited settings, can be problematic; dried blood spots (DBS)-EFV measurements thus provide a cheap easy alternative for therapeutic drug monitoring. Our aim was to develop and validate a liquid chromatography-mass spectrometry method to quantify EFV in DBS collected as part of clinical trials in resource-limited settings.

METHODS: DBS for standards, quality control samples, and patient samples were excised and then extracted with ethyl acetate/n-hexane (50/50 vol/vol) after addition of internal standard hexobarbital, and 1 mol/L K2CO3. The extract was evaporated to dryness, the residue reconstituted in mobile phase and analyzed directly by liquid chromatography-mass spectrometry. Gradient elution was on a reverse-phase C18 column using 1 mmol/L ammonium acetate in water and acetonitrile. Quantification was by selected reaction monitoring in negative ionization mode. DBS samples were obtained at several time points over 24 hours from HIV+ patients on either 400 or 600 mg EFV in combination with emtricitabine/tenofovir.

RESULTS: The internal standard and EFV eluted at 2.68 and 3.54 minutes, respectively in a 5-minute run time. Matrix effects were minimal (-5.4%). Calibration curves were validated over a concentration range of 25-5000 ng/mL. Intra-assay and interassay variations ranged between 6.7% and 8.7% for imprecision and 100.3% and 104.2% for accuracy. Mean recovery was >64%. The DBS data showed a strong positive correlation with a validated plasma EFV assay (R = 0.9764, P < 0.001). EFV concentrations from DBS were approximately 42% lower than the paired plasma values, and the ratio of blood/plasma did not change over the dosing interval.

CONCLUSIONS: The validated assay is now routinely applied to clinical samples measuring DBS EFV for pharmacokinetic analysis. The methodology is robust, accurate, and sensitive.

OBJECTIVES: The validation and clinical application of an LC-MS/MS method for the quantification of nevirapine in dried blood spots (DBS) and dried breast-milk spots (DBMS) are presented.

METHODS: DBS and DBMS were prepared from 50 and 30 μL of nevirapine-spiked whole blood and human breast milk, respectively. Chromatographic separation was achieved on a reverse-phase C18 column with 0.1% formic acid in water/acetonitrile using a solvent gradient programme at a flow rate of 400 μL/min, and detection was by a TSQ Quantum Access triple quadrupole mass spectrometer. The clinical application was evaluated in HIV-positive nursing mothers and their breastfed infants.

RESULTS: The assay was validated over the concentration range 50-10,000 ng/mL. Accuracy ranged from 93.3% to 113.4% and precision ranged from 1.9% to 12.0%. The mean (percentage coefficient of variation) recovery of nevirapine from DBS and DBMS was ≥ 70.7% (≤ 8.2) and the matrix effect was ≤ 1.04 (≤ 6.1). Nevirapine was stable in DBS and DBMS for ≥ 15 months at room temperature and -80°C. Mean (SD) AUC0-12, Cmax and Cmin in maternal plasma versus breast milk were 57,808 ng · h/mL (24,315) versus 55,817 ng · h/mL (22,368), 6140 ng/mL (2605) versus 5231 ng/mL (2215) and 4334 ng/mL (1880) versus 4342 ng/mL (2245), respectively. The milk-to-plasma concentration ratio over the dosing interval was 0.94 (0.15). Infant plasma concentrations 2 and 8 h after maternal dosing were 580.6 ng/mL (464.7-1607) and 584.1 ng/mL (381.5-1570), respectively.

CONCLUSIONS: These methods further extend opportunities for conducting clinical pharmacokinetic studies in nursing mother-infant pairs, especially in resource-limited settings.

Objectives The validation and clinical application of an LC–MS/MS method for the quantification of nevirapine in dried blood spots (DBS) and dried breast-milk spots (DBMS) are presented.

Methods DBS and DBMS were prepared from 50 and 30 μL of nevirapine-spiked whole blood and human breast milk, respectively. Chromatographic separation was achieved on a reverse-phase C18 column with 0.1% formic acid in water/acetonitrile using a solvent gradient programme at a flow rate of 400 μL/min, and detection was by a TSQ Quantum Access triple quadrupole mass spectrometer. The clinical application was evaluated in HIV-positive nursing mothers and their breastfed infants.

Results The assay was validated over the concentration range 50–10 000 ng/mL. Accuracy ranged from 93.3% to 113.4% and precision ranged from 1.9% to 12.0%. The mean (percentage coefficient of variation) recovery of nevirapine from DBS and DBMS was ≥70.7% (≤8.2) and the matrix effect was ≤1.04 (≤6.1). Nevirapine was stable in DBS and DBMS for ≥15 months at room temperature and −80°C. Mean (SD) AUC0–12, Cmax and Cmin in maternal plasma versus breast milk were 57 808 ng·h/mL (24 315) versus 55 817 ng·h/mL (22 368), 6140 ng/mL (2605) versus 5231 ng/mL (2215) and 4334 ng/mL (1880) versus 4342 ng/mL (2245), respectively. The milk-to-plasma concentration ratio over the dosing interval was 0.94 (0.15). Infant plasma concentrations 2 and 8 h after maternal dosing were 580.6 ng/mL (464.7–1607) and 584.1 ng/mL (381.5–1570), respectively.

Conclusions These methods further extend opportunities for conducting clinical pharmacokinetic studies in nursing mother–infant pairs, especially in resource-limited settings.

The aims of this study were to determine the sensitivity, specificity, positive and negative predictive values of ultrasonography in detecting zygomaticomaxillary complex fractures, and to highlight factors that may affect the validity of ultrasonography in the diagnosis of zygomaticomaxillary complex fracture. Twenty-one patients with suspected fractures of the zygomaticomaxillary complex presenting at the authors' hospital were included in this prospective study. All the patients had plain radiographic and computed tomography (CT) investigations. All underwent ultrasonographic examination of the affected region using an ultrasound machine with a 7.5 MHz probe. The different radiologists were not aware of the results of the other two investigations. Statistical significance was inferred at P<0.05. The validity of ultrasonography varied with fracture sites with a sensitivity of 100% for zygomatic arch fractures, 90% for infraorbital margin fractures and 25% for frontozygomatic suture separation. Specificity was 100% for the three types of fracture. There was no statistically significant difference in the ability of CT scan and ultrasonography to diagnose fractures from various zygomaticomaxillary complex fracture sites (P=0.47). Ultrasonography has proved to be a valid tool for the diagnosis of zygomatic arch and displaced infraorbital margin fractures.

The links between climate issues and sustainable development are manifold. Given these interconnections, the lack of close integration of the sustainable development and climate change literatures is puzzling; part of the reason for this lack of connectivity may be the very different research and policy traditions out of which each field developed. This paper argues that integrating climate change and sustainable development approaches, concepts and methods may have some important benefits. To demonstrate this point, we briefly discuss recent developments in both the climate change and sustainable development fields and then turn to the question of how to integrate them. The analysis suggests several conclusions of possible relevance to climate change and sustainable development research, including the need for an approach to scenario analysis that integrates across all aspects of climate change and sustainable development research, and the critical importance of alternative development paths and the assumptions about the reference case or baseline that underlie any analysis.

Removal of vegetation to give space for urban expansion might result in the temperature rise in cities. The present study compares the LSTs derived from Moderate Resolution Imaging Spectroradiometer (MODIS) with observed air temperature from ground weather data. The natures of the materials that are usually found in the urban area are typically concrete and asphalt materials which affect the urban atmospheric system. In this study, variation in urban land surface temperatures (LST) using MODIS and in-situ meteorological data were examined. MODIS data and daily rainfall, minimum (Tm) and maximum (Tmx) temperature from ground weather station were used. The results reveal that average LSTs during the dry season are noticeably higher for both daytime during November: 34.62 °C, December: 33.75 °C, January: 34.68 °C, February: 35.02 °C and March: 34.87 °C. There are notable differences in the LST observed between daytime and nighttime for both MOD11A2 and MYD11A2 and that of maximum and minimum air temperature from in-situ meteorological data. MOD11A2 is a better proxy for daily maximum and minimum air temperature than MYD11A2, though seasonal variations in the extent of LST occurs during the wet and dry season. The study shows that the contribution of the urban LSTs was comparatively smaller at night than the day, perhaps as result of the variations in the amount of solar radiation received by the day and night times.