Sensitive, high throughput HPLC-MS/MS method with on-line sample clean-up for everolimus measurement

A new HPLC-MS/MS method for everolimus measurement was developed that includes the following features: small sample volume, short run time, fast, simple and cost-efficient sample preparation, assessment of performance of two internal standards (IS), SDZ RAD 223-756 and ascomycin and comparison of the method with an HPLC-MS/MS reference method. The authors established a multiple reaction monitoring positive ion HPLC-MS/MS method with on-line extraction and sample cleanup. This procedure includes: an API 2000 triple quadrupole mass spectrometer with turbo-ion spray, built-in Valco switching valve, an HPLC system; guard column; a Nova-Pak C18 analytical column; washing solution, methanol:30 mM ammonium acetate pH 5.1 (80:20); eluting solution, methanol:30 mM ammonium acetate pH 5.1 (97:3); flow rate 0.8 mL/min; and a run time of 2.8 minutes. The first and third quadrupoles were set to detect the ammonium adduct ion and a high mass fragment of everolimus (m/z 975.5-->908.5), and two ISs: SDZ RAD 223-756 (m/z 989.8-->922.8) and ascomycin (m/z 809.5-->756.5). The LLOQ was 1.0 microg/L for everolimus using either IS. Between day precision ranged from 3.1% to 5.7% for SDZ RAD 223-756 and 6.0% to 8.6% for ascomycin using spiked blood with everolimus concentrations 2.0 to 25.0 microg/L. Absolute recoveries using spiked samples over the range of 2.5 to 25 mug/L averaged 77.3% (SDZ RAD 223-756) and 76.8% (ascomycin). No matrix effect on everolimus was demonstrated based on the mean observed signal detection of 98.6% (SDZ RAD 223-756) and 105% (ascomycin). Comparison of everolimus concentrations obtained using this method with two internal standards with a reference laboratory demonstrated that the mean everolimus concentration obtained with ascomycin was statistically different (lower) than results with the reference method and the method that used SDZ RAD 223-756 as the internal standard gave equivalent results compared with the reference method.     

Development and evaluation of clinical reasoning using ‘think aloud’ approach in pharmacy undergraduates – A mixed-methods study

IntroductionGiven the widespread use of clinical reasoning (CR) in the healthcare practice, it is essential to inculcate the CR practice in undergraduate pharmacy education which can not only facilitate their clinical education and clinical rotations but can also help them become better clinical pharmacists. There is very limited CR employed in the pharmacy curriculum and practice in the Middle East countries. This study aimed to develop and evaluate CR practice in pharmacy undergraduates in one college of pharmacy in Saudi Arabia.MethodsWe employed a mixed-methods methodology that included two phases. In Phase I, students were introduced to CR practice (‘think aloud’ method) and given geriatric clinical cases which they used in two sessions together with a tutor. This was followed by the writing of SOAP notes using the tutor feedback and completion of a survey that included a self-reflection about their experience of using the CR method. Phase II included face-to-face semi-structured interviews involving selected students that were recruited via convenience sampling to further explore the issues identified in Phase I of the study.ResultsOf the 155 students who completed the survey (response rate 94%), the majority of them agreed that CR using the ‘think aloud’ method was useful in gathering (92%) and interpreting (95%) relevant patient information, identifying medication-related problems (95%), exploring therapeutic options for the problem(s) (93%) and formulating a treatment plan for the patient (90%). Qualitative data analysis of the 12 interviews was consistent with these findings. Furthermore, it provided an insight into the challenges faced by the students in applying this CR method.ConclusionsStudents found the practice of CR using the ‘think aloud’ method helpful in working through given cases and taking clinical decisions. This method can be widely employed in pharmacy education and practice.     

Development and evaluation of clinical reasoning using ‘think aloud’ approach in pharmacy undergraduates – A mixed-methods study

IntroductionGiven the widespread use of clinical reasoning (CR) in the healthcare practice, it is essential to inculcate the CR practice in undergraduate pharmacy education which can not only facilitate their clinical education and clinical rotations but can also help them become better clinical pharmacists. There is very limited CR employed in the pharmacy curriculum and practice in the Middle East countries. This study aimed to develop and evaluate CR practice in pharmacy undergraduates in one college of pharmacy in Saudi Arabia.MethodsWe employed a mixed-methods methodology that included two phases. In Phase I, students were introduced to CR practice (‘think aloud’ method) and given geriatric clinical cases which they used in two sessions together with a tutor. This was followed by the writing of SOAP notes using the tutor feedback and completion of a survey that included a self-reflection about their experience of using the CR method. Phase II included face-to-face semi-structured interviews involving selected students that were recruited via convenience sampling to further explore the issues identified in Phase I of the study.ResultsOf the 155 students who completed the survey (response rate 94%), the majority of them agreed that CR using the ‘think aloud’ method was useful in gathering (92%) and interpreting (95%) relevant patient information, identifying medication-related problems (95%), exploring therapeutic options for the problem(s) (93%) and formulating a treatment plan for the patient (90%). Qualitative data analysis of the 12 interviews was consistent with these findings. Furthermore, it provided an insight into the challenges faced by the students in applying this CR method.ConclusionsStudents found the practice of CR using the ‘think aloud’ method helpful in working through given cases and taking clinical decisions. This method can be widely employed in pharmacy education and practice.     

Individualizing theophylline dosage: Evaluation of a single-point maintenance dose prediction method

Summary A dosage prediction method to estimate theophylline clearance and dose requirement was evaluated in 22 outpatients with partly reversible obstructive airways disease. The steady state theophylline dose required to achieve a target concentration (Css) was predicted using a single serum theophylline determination 8 h after a single oral test dose. In 17 nonsmoking patients a mean absolute deviation of 8.2% (range 0.0–21.7%) between predicted and observed Css was found, and in 5 smoking patients the mean deviation was 34.0% (range 2.2–53.8%). In 17 healthy smokers the single-point method was found to predict theophylline clearance at a sampling time of 8 h with a prediction error of 11.3 (range 0.8–25.3%) compared to the clearance determination using the area under the curve. In addition, a numerical simulation program to assess the influence of absorption, elimination and sampling time on predictive accuracy showed that the method could be successfully applied to a patient population with elimination rate constants between 0.07 1/h and 0.25 1/h, allowing a mean prediction error of 15%.     

A rapid non‐target screening method for determining prohibited substances in human urine using liquid chromatography/high‐resolution tandem mass spectrometry

Target analysis using liquid chromatography–tandem mass spectrometry is applied for rapidly detecting various prohibited doping substances. Frequent modification is required as additional substances are prohibited. We developed and validated a non‐target screening method requiring no further modification because it analyzes the full spectrum of data in fixed m/z ranges. Urine samples were extracted using solid‐phase extraction and analyzed by employing a method that combines full scan and variable data independent acquisition using high‐resolution mass spectrometry; and all prohibited substances in the urine samples were successfully detected using our screening method. The method was validated in terms of specificity (no interferences), recoveries (29%–131%), matrix effects (35%–237%), limites of detection (0.0002–100 ng/mL), and intra‐ and inter‐day precisions (coefficients of variation lower than 25%). The applicability of this method to doping tests was evaluated by analyzing 14 urine samples. As a result, the non‐target screening method is efficient for conducting anti‐doping tests because it can be applied without any further modification to prohibited drugs as well as to unknown targets that can be prohibited in the future.     

Prediction Methods for Nicotine Clearance Using Cotinine and 3-Hydroxy-Cotinine Spot Saliva Samples II. Model Application

To develop and compare methods that predict individual nicotine (NIC) clearance, which reflects CYP2A6 activity, using random saliva cotinine (COT) and trans 3′-hydroxycotinine (3HC) measurements. COT and 3HC saliva concentrations in smokers were simulated utilizing a mechanistic population pharmacokinetic model of NIC metabolism that was adapted from the one described in a companion paper. Four methods to predict NIC clearance using the metabolites concentrations were compared. The precision bias, and the fraction of predictions that are made with an absolute error below 25% were the performance measures evaluated. Four prediction methods were compared: (M1) reference method, an intercept slope model of the metabolite concentration ratios ([3HC]/[COT]) (M2) an intercept slope model of the natural logarithm of the metabolite ratios (M3) a spline of the logarithm of the metabolite ratios (M4) Maximal Posteriori Bayesian estimate of NIC clearance conditioned on the model, COT and 3HC concentrations. In addition, the effect of smoking patterns on the concentrations of COT and 3HC was evaluated. The precision, accuracy, and the fraction of predictions with an absolute error below 25%, were higher for methods M2–M4 compared to method M1. However, the differences between M2 and M4 were small. Additionally, smoking pattern did not affect the metabolite concentration profiles. Predicting NIC clearance using an intercept slope model of the natural logarithm of the ratio of 3HC to COT appears to be a relatively simple method that is better than using the metabolite ratio directly. This method has a bias of approximately −10%, precision of approximately 60%. The fraction of estimates below an absolute error of 25% is 43%. These results support use of M2 to estimate CYP2A6 activity in smokers in the clinical setting.     

高效液相色谱法测定人血浆中伏立康唑浓度

目的：建立一种简单高效的高效液相色谱（HPLC）法用来检测人体中伏立康唑的血药浓度，并应用于临床中伏立康唑用药监测，以促进其个体化用药。方法：色谱柱：Kromasil C₁₈（4.6 mm×150 mm，5 μm），柱温：35℃，流速：1.0 mL·min^-1，流动相：甲醇-水（60：40），检测波长：257 nm，内标：酮康唑。对该方法进行方法学验证。结果：该方法专属性良好，血浆中伏立康唑在0.1~20.0 μg·mL^-1范围内线性良好（r=0.999 6），定量下限为0.1 μg·mL^-1。高、中、低3个浓度提取回收率分别为（90.68±10.32）%、（92.82±8.26）%、（97.47±4.58）%；日内精密度RSD分别为5.87%、7.85%、4.10%；日间精密度RSD分别为5.64%、3.30%、2.74%。对某院20例（男12例，女8例）使用伏立康唑抗真菌治疗的患者运用该方法进行了监测，结果显示浓度范围在0.71~13.51 μg·mL^-1之间。结论：本方法专属性高，操作简便，结果准确，可用于临床上伏立康唑血药浓度的检测，从而促进其个体化用药的推广。     

Micromeritics and release behaviours of cellulose acetate butyrate microspheres containing theophylline prepared by emulsion solvent evaporation and emulsion non-solvent addition method

The present research work compares the effect of microsphere preparation technique on micromeritics and release behaviors of theophylline microspheres. Microspheres were prepared by oil-in oil (O₁/O₂) emulsion solvent evaporation method (ESE) using different ratios of anhydrous theophylline to cellulose acetate butyrate (CAB). Cyclohexane was used as non-solvent to modify the ESE technique (MESE method) and the effect of non-solvent volume on properties of microspheres was investigated. The obtained microspheres were analyzed in terms of drug content, particle size and encapsulation efficiency. The morphology of microsphere was studied using scanning electron microscope. The solid state of microspheres, theophylline and CAB were investigated using X-ray, FT-IR and DSC. The drug content of microspheres prepared by MESE method was significantly lower (15.54% ± 0.46) than microspheres prepared by ESE method (41.08 ± 0.40%). The results showed that as the amount of cyclohexane was increased from 2 mL to 6 mL the drug content of microspheres was increased from 15.54% to 28.71%. Higher encapsulation efficiencies were obtained for microspheres prepared by ESE method (95.87%) in comparison with MESE method (64.71%). Mean particle size of microsphere prepared by ESE method was not remarkably affected by drug to polymer ratio, whereas in MSES method when the volume of cyclohexane was increased the mean particle size of microsphere was significantly decreased. The ratio of drug to polymer significantly changed the rate of drug release from microspheres and the highest drug release was obtained for the microsphere with high drug to polymer ratio. The amount of cyclohexane did not significantly change the drug release. Although, x-ray showed a small change in crystallinity of theophylline in microspheres, DSC results proved that theophylline in microspheres is in amorphous state. No major chemical interaction between the drug and polymer was reported during the encapsulation process.     

Comparison of Patient Simulation Methods Used in a Physical Assessment Course

Objective. To determine whether there is a difference in student pharmacists’ learning or satisfaction when standardized patients or manikins are used to teach physical assessment.Design. Third-year student pharmacists were randomized to learn physical assessment (cardiac and pulmonary examinations) using either a standardized patient or a manikin.Assessment. Performance scores on the final examination and satisfaction with the learning method were compared between groups. Eighty and 74 student pharmacists completed the cardiac and pulmonary examinations, respectively. There was no difference in performance scores between student pharmacists who were trained using manikins vs standardized patients (93.8% vs. 93.5%, p=0.81). Student pharmacists who were trained using manikins indicated that they would have probably learned to perform cardiac and pulmonary examinations better had they been taught using standardized patients (p<0.001) and that they were less satisfied with their method of learning (p=0.04).Conclusions. Training using standardized patients and manikins are equally effective methods of learning physical assessment, but student pharmacists preferred using standardized patients.     

Improved 68Ga‐labeling method using ethanol addition: Application to the α‐helical peptide DOTA‐FAMP

We examined the ⁶⁸Ga labeling of the α‐helical peptide, DOTA‐FAMP, and evaluated conformational changes during radiolabeling. ⁶⁸Ga‐DOTA‐FAMP is a positron emission tomography probe candidate for atherosclerotic plaques. The labeling yield achieved by Zhernosekov's method (using acetone for ⁶⁸Ga purification) was compared with that achieved by the original and 2 modified Mueller's methods (using NaCl solution). Modified method I involves desalting the ⁶⁸Ga prior to labeling, and modified method II involves the inclusion of ethanol in the labeling solution. The labeling yield using Zhernosekov's method was 62% ± 5.4%. In comparison, Mueller's original method gave 8.9% ± 1.7%. Modified method I gave a slight improvement of 32% ± 2.1%. Modified method II further increased the yield to 66% ± 3.4%. Conformational changes were determined by circular dichroism spectroscopy, revealing that these differences could be attributed to conformational changes. Heat treatment affects peptide conformation, which leads to aggregation and decreases the labeling yield. Mueller's method is simpler, but harsh conditions preclude its application to biomolecules. To suppress aggregation, we included a desalting process and added ethanol in the labeling solution. These changes significantly improved the labeling yield. Before use for imaging, conformational changes of biomolecules during radiolabeling should be evaluated by circular dichroism spectroscopy to ensure the homogeneity of the labeled product.     

Development and evaluation of febuxostat solid dispersion through screening method

Febuxostat (Febux) is a BCS II drug and has a very low solubility. In order to overcome this shortcoming, the purpose of study is to increase the in vitro dissolution (%) and drug release (%) of Febux by using a screening method. The Febux-SD formulation was prepared by screening solubilizers, pH agents, and carriers using with a solvent evaporation method.The novel Febux SD formulation was successfully developed. The dissolution (%) of Febux of optimal formulation (SD3) was higher than that of Feburic® tab in pH 1.2, distilled water (DW), and pH 6.8 buffer by 6.3-, 2.6-, and 1.1-fold, respectively, at 60 min. The in vitro drug release (%) and permeability (μg/cm²) of SD3 formulation were improved compared to those of Feburic® tab in the pH shifting method and PBS (7.4), respectively. The SD3 formulation was well maintained the stability for 6 months, and that of physicochemical properties were altered. In conclusion, the Febux solubilization study with meglumine was first attempted and successfully performed. Through the improved dissolution (%) of Febux, high bioavailability of SD3 formulation is expected in animal and human studies.     

α-Tocopherol,Flavonoid, and Phenol Contents and Antioxidant Activity of Ficus carica. Leaves

Abstract

The aim of the current study was to evaluate the α.-tocopherol content and to investigate the antioxidant capacities of the extracts prepared from the leaves of Ficus carica. L. (Moraceae). The antioxidant capacities of the extracts were evaluated by the phosphomolibdenum spectrophotometric method. α.-Tocopherol content was determined by using a high-performance liquid chromatography (HPLC)-UV method. Total flavonoid content was determined by using the aluminium chloride method. Total phenol content was estimated by a modified colorimetric method using Folin-Ciocalteau reagent. The results clearly demonstrate that these extracts have antioxidant capacity. Antioxidant capacity results are consistent with total flavonoid and phenol contents. The α.-tocopherol content of the n.-hexane extract was found to be 3.2788%, whereas it was calculated as 0.0570% on the dry-weight basis of the leaves.     

Quantification of levetiracetam in human plasma by liquid chromatography-tandem mass spectrometry: application to therapeutic drug monitoring

A rapid, selective, reliable, precise, accurate, and reproducible tandem mass spectrometric (MS-MS) method for the quantification of levetiracetam (LEV) in human plasma using adenosine as an internal standard (IS) has been developed and validated. The drug and IS were extracted by solid phase extraction (SPE) technique and analyzed on Symmetry((R)) C(18) column (5 microm, 3.9 mm x 50 mm) using a mobile phase of methanol-water-formic acid (97:03:0.25, v/v/v) at a flow rate of 0.2 ml/min. Quantitation was achieved using a positive electrospray ionization (ESI+) interface employing multiple reaction monitoring (MRM) mode at MRM transitions m/z 171>126 and m/z 268>136 for LEV and IS, respectively. The method was validated over the concentration range of 1.0-40 microg/ml (r>0.99) with a limit of quantification of 1.0 microg/ml (R.S.D.%; 4.1 and Bias%; -9.0 to + 11.0%). Intra- and inter-run precision of LEV assay at three concentrations ranged from 0.6 to 8.9% with accuracy (bias) varied from -4.0 to 8.6% indicating good precision and accuracy. Analytical recoveries of LEV and IS from spiked human plasma were in the range of 91.7-93.4% and 80.2-84.1%, respectively. Stability of LEV in human plasma samples at different conditions showed that the drug was stable under the studied conditions. Matrix effect study showed a lack of matrix effect on mass ions of LEV and IS. The described method compared well with the commercial HPLC-UV method of Chromsystem (r(2)=0.99). The suitability of the developed method for therapeutic drug monitoring was demonstrated by measuring LEV in human plasma samples of epileptic patients treated with LEV.     

Comparative evaluation of tableting compression behaviors by methods of internal and external lubricant addition: Inhibition of enzymatic activity of trypsin preparation by using external lubricant addition during the tableting compression process

This study evaluated tableting compression by using internal and external lubricant addition. The effect of lubricant addition on the enzymatic activity of trypsin, which was used as a model drug during the tableting compression process, was also investigated. The powder mixture (2% crystalline trypsin, 58% crystalline lactose, and 40% microcrystalline cellulose) was kneaded with 5% hydroxypropyl cellulose aqueous solution and then granulated using an extruding granulator equipped with a 0.5-mm mesh screen at 20 rpm. After drying, the sample granules were passed through a 10-mesh screen (1680 μm). A 200-mg sample was compressed by using 8-mm punches and dies at 49, 98, 196, or 388 MPa (Mega Pascal) at a speed of 25 mm/min. The external lubricant compression was performed using granules without lubricant in the punches and dies. The granules were already dry coated by the lubricant. In contrast, the internal lubricant compression was performed using sample granules (without dry coating) containing 0.5% lubricant. At 98 MPa, for example, the compression level using the external lubricant addition method was about 13% higher than that for internal addition. The significantly higher compressing energy was also observed at other MPas. By comparison, the friction energy for the external addition method calculated based on upper and lower compression forces was only slightly larger. The hardness of tablets prepared using the internal addition method was 34% to 48% lower than that for the external addition method. The total pore volume of the tablet prepared using the external addition method was significantly higher. The maximum ejection pressure using the no-addition method (ie, the tablet was prepared using neither dry-coated granules nor added lubricant) was significantly higher than that of other addition methods. The order was as follows: no addition, external addition, and then internal addition. The ejection energy (EE) for internal addition was the lowest; for no addition, EE was the highest. In the dissolution test, the tablets obtained using external addition immediately disintegrated and showed faster drug release than those prepared using internal addition. This result occurred because the water penetration rate of the tablet using the external addition was much higher. The trypsin activity in tablets prepared using the external addition method was significantly higher than that produced using the internal addition method at the same pressure. All these results suggest that the external addition method might produce a fast-dissolution tablet. Because the drug will be compressed using low pressure only, an unstable bulk drug may be tableted without losing potency.     

Gas chromatographic method for determining very long chain fatty acids that compose D003 in aqueous suspensions from 20 to 200 mg/ml,used in pharmacological and toxicological studies

D003 is a natural mixture of fatty acids (C(24:0) to C(36:0)), which shows antiplatelet, antithrombotic, and cholesterol-lowering effects in experimental models. A specific gas chromatographic method, using a BPX-5 wide-bore column and 1-nonadecanoic acid as internal standard, was developed and validated to determine the content of D003 in 20-200 mg/ml aqueous suspensions, which are used in pharmacological and toxicological studies. Fatty acids were extracted with chloroform and converted to methyl esters derivatives using 5% aqueous HCl-methanol. The method was linear for suspensions ranging from 10 to 250 mg/ml (correlation coefficient=0.9998) and showed a good accuracy, with average recoveries (98.5-101.28%) no significantly different from 100%, according to the Student t-test (P=0.05). The RSDs were <2.2%, indicating that the method has a good repeatability. The intermediate precision was good too, with RSDs between 1.20 and 2.10%. This method is suitable for quality control of these suspensions.     

Quantitative analysis of buprenorphine and norbuprenorphine in urine using liquid chromatography tandem mass spectrometry

Buprenorphine is an opioid analgesic drug that is used as an alternative to methadone to treat heroin addiction. Established methods for the analysis of buprenorphine and its metabolites in urine such as gas chromatography-mass spectrometry (GC-MS) involve complicated sample extraction procedures. The aim of the present study was to develop a sensitive yet straightforward method for the simultaneous analysis of buprenorphine and norbuprenorphine in urine using liquid chromatography-MS-MS. The method comprised an enzymatic hydrolysis using Patella vulgata b-glucuronidase, followed by centrifugation and direct analysis of the supernatant. The limits of detection and quantitation were < 1 microg/L for buprenorphine and < 1 and 4 microg/L, respectively, for norbuprenorphine. Assay coefficients of variation (CVs) were < 15%, with the exception of concentrations close to the limit of quantitation, where CVs were below 20%. In direct comparison with an established GC-MS protocol, the method showed minimal negative bias (8.7% for buprenorphine and 1.8% for norbuprenorphine) and was less susceptible to sample carryover. The extent of conjugation in unhydrolyzed urine was investigated and found to be highly variable, with proportions of unconjugated buprenorphine and norbuprenorphine of 6.4% [range 0% to 67%; standard deviation (SD) 9.7%] and 34% (range 0% to 100%; SD 23.8%), respectively.     

An ecofriendly agar extraction strategy using KOH for boiling extraction and MgCl2 for alkali neutralization

In this study, a new strategy for extracting agar from Gracilaria was proposed to replace the traditional NaOH extraction, using low concentration of KOH solution and MgCl₂ neutralization (KOH–MgCl₂ method) under homogeneous conditions. The KOH–MgCl₂ method was more convenient than the traditional NaOH method, with 84% less alkali consumption, 76.4% less water consumption, and 80% less wastewater. The physicochemical properties of the agar extracted by the KOH–MgCl₂ method (KMA) at different alkali concentrations were compared with those of the agar extracted with the traditional NaOH extraction method (NA) under scale-up tests. The results showed that KMA₃ had lower sulfate content and higher 3,6-anhydro-α-l-galactose content and molecular weight than NA, indicating that the KOH–MgCl₂ method had better desulfurization and lower degradation effect. Thus, this new method has potential to replace the traditional NaOH extraction method.     

A qPCR Assay to Detect and Quantify Shiga Toxin-Producing E. coli (STEC) in Cattle and on Farms: A Potential Predictive Tool for STEC Culture-Positive Farms

Shiga toxin-producing E. coli (STEC), of various serogroups harboring the intimin gene, form a serious threat to human health. They are asymptomatically carried by cattle. In this study, a quantitative real-time PCR (qPCR) method was developed as a molecular method to detect and quantify Shiga toxin genes stx1 and stx2 and the intimin gene eae. Subsequently, 59 fecal samples from six farms were tested using qPCR and a culture method as a reference. Three farms had contaminated animals as demonstrated by the culture method. Culture-positive farms showed moderate significantly higher stx prevalences than culture-negative farms (p = 0.05). This is the first study which showed preliminary results that qPCR can predict STEC farm contamination, with a specificity of 77% and a sensitivity of 83%, as compared with the culture method. Furthermore, the presence or quantity of stx genes in feces was not correlated to the isolation of STEC from the individual animal. Quantitative data thus did not add value to the results. Finally, the detection of both stx and eae genes within the same fecal sample or farm using qPCR was not correlated with the isolation of an eae-harboring STEC strain from the respective sample or farm using the culture method.