Use of qNMR to determine HPLC relative response factors for botanical reference standards used in pharmacopeial monographs.

In modern botanical pharmacopeial monographs, one measurement of content is the quantitation of relevant constituents as marker compounds. The use of suitable reference standards (RSs) to quantify multiple compounds by HPLC is recommended in the U.S. Pharmacopeial (USP) botanical monographs. However, these substances may be expensive and difficult to develop into an RS. Surrogate RSs could be used instead of the actual constituents, provided that the relative response factors (RRFs) of each analyte to the selected surrogate RS are known. USP monographs of both Sichuan Lovage Rhizome and Dong Quai Root recognize Z-ligustilide as a major characteristic marker compound, making quantitation of Z-ligustilide and its analog(s) relevant for quality control. However, because Z-ligustilide is unstable, it is difficult to develop it into a quantitative RS. Instead, oxybenzone was selected as a surrogate external quantitative RS because of its similar chromatographic behavior to Z-ligustilide, its stability, and affordable cost. The RRF determination of Z-ligustilide to oxybenzone by the conventional HPLC procedure is challenging due to both the instability of the purified Z-ligustilide at ambient temperature and the difficulty of determining its purity. Therefore, a qNMR method was used to overcome these challenges as it enables to directly measure the mass ratio of Z-ligustilide to oxybenzone in the stock solution without the need for weighing and purity information. In the present study, RRF values of 1.01, 0.46, and 0.89 for Z-ligustilide, senkyunolide A, and ferulic acid relative to oxybenzone, respectively, were determined using the qNMR-based methodology.

Stability-Indicating Method Development and Validation for Busulfan Drug Substance by Gas Chromatography-Flame Ionization Detector.

A new, simple and stability-indicating gas chromatography-flame ionization detection (GC-FID) method was developed and validated for the quantitative determination of busulfan and its organic impurities (OI) in drug substance without derivatization. The chromatographic attributes were achieved on a fused silica capillary column (0.53 mm × 30 m, 1.0 µm, USP Phase G42), using hydrogen as a carrier gas with a split ratio of 1:1. Forced degradation studies were conducted to establish the stability-indicating capability and method specificity that showed the stressed busulfan peak was free from any co-elution. Robustness study demonstrated the chromatograms remained mostly unaffected under deliberate, but small variations of chromatographic parameters, establishing the reliability of the method during routine usage. The method was shown to be reliable, sensitive, specific, linear, accurate, precise and rugged in the 1,4-butanediol concentration range of 1-20 µg/mL. The method, intended for compendial uses, is suitable for quantitative analysis of busulfan and its organic impurities in drug substances.

Clarification of the USP compendial procedure for phenoxybenzamine hydrochloride via updating impurity profiles.

The current United States Pharmacopeia-National Formulary (USP-NF) and the British Pharmacopoeia phenoxybenzamine (PBA) hydrochloride drug substance and drug product monographs describe an HPLC procedure for the determination of a specified impurity "tertiary amine phenoxybenzamine" and use the resolution of an "unknown related substance" from PBA as a system suitability criterion; however, neither structural information of the "unknown related substance" is provided nor reference standards of the two impurities are available. The ambiguity in pharmacopeias poses difficulties in implementing the procedure for quality control. To clarify the degradation pathways, and incorporate the impurity profile of PBA into the USP monographs, the degradation of PBA was revisited. PBA undergoes rapid degradation in neutral or basic aqueous solutions to generate the "tertiary amine phenoxybenzamine" as the predominant degradation product, which was confirmed as phenoxybenzamine hydroxide (PBA-OH). In addition, the "unknown related substance" was proposed as the phenoxybenzamine nitrile (PBA-CN) on the basis of LC-MS studies. The identity of PBA-CN was unambiguously verified via chemical synthesis, HPLC and NMR analyses. A stability-indicating method was developed and validated for the determination of PBA and its impurities, and was used to support USP monograph modernization.

Quantitative analysis of 3-isopropylamino-1,2-propanediol as a degradation product of metoprolol in pharmaceutical dosage forms by HILIC-CAD.

Aryloxypropanolamine is an essential structural scaffold for a variety of ß-adrenergic receptor antagonists such as metoprolol. Molecules with such a structural motif tend to degrade into α, ß-hydroxypropanolamine impurities via a radical-initiated oxidation pathway. These impurities are typically polar and nonchromophoric, and are thus often overlooked using traditional reversed phase chromatography and UV detection. In this work, stress testing of metoprolol confirmed the generation of 3-isopropylamino-1,2-propanediol as a degradation product, which is a specified impurity of metoprolol in the European Pharmacopoeia (impurity N). To ensure the safety and quality of metoprolol drug products, hydrophilic interaction chromatography (HILIC) methods using Halo Penta HILIC column (150 mm × 4.6 mm, 5 µm) coupled with charged aerosol detection (CAD) were developed and optimized for the separation and quantitation of metoprolol impurity N in metoprolol drug products including metoprolol tartrate injection, metoprolol tartrate tablets, and metoprolol succinate extended-release tablets. These HILIC-CAD methods were validated per USP validation guidelines with respect to specificity, linearity, accuracy, and precision, and have been successfully applied to determine impurity N in metoprolol drug products.

Structural confirmation of sulconazole sulfoxide as the primary degradation product of sulconazole nitrate.

Sulconazole has been reported to degrade into sulconazole sulfoxide via sulfur oxidation; however, structural characterization data was lacking and the potential formation of an N-oxide or sulfone could not be excluded. To clarify the degradation pathways and incorporate the impurity profile of sulconazole into the United States Pharmacopeia-National Formulary (USP-NF) monographs, a multifaceted approach was utilized to confirm the identity of the degradant. The approach combines stress testing of sulconazole nitrate, chemical synthesis of the degradant via a hydrogen peroxide-mediated oxidation reaction, semi-preparative HPLC purification, and structural elucidation by LC-MS/MS and NMR spectroscopy. Structural determination was primarily based on the comparison of spectroscopic data of sulconazole and the oxidative degradant. The mass spectrometric data have revealed a McLafferty-type rearrangement as the characteristic fragmentation pathway for alkyl sulfoxides with a ß-hydrogen atom, and was used to distinguish the sulfoxide from N-oxide or sulfone derivatives. Moreover, the generated sulconazole sulfoxide was utilized as reference material for compendial procedure development and validation, which provides support for USP monograph modernization.

Development and validation of a hydrophilic interaction chromatography method coupled with a charged aerosol detector for quantitative analysis of nonchromophoric α-hydroxyamines, organic impurities of metoprolol.

The European Pharmacopeia (EP) metoprolol impurities M and N are polar, nonchromophoric α-hydroxyamines, which are poorly retained in a conventional reversed-phase chromatographic system and are invisible for UV detection. Impurities M and N are currently analyzed by TLC methods in the EP as specified impurities and in the United States Pharmacopeia-National Formulary (USP-NF) as unspecified impurities. In order to modernize the USP monographs of metoprolol drug substances and related drug products, a hydrophilic interaction chromatography (HILIC) method coupled with a charged aerosol detector (CAD) was explored for the analysis of the two impurities. A comprehensive column screening that covers a variety of HILIC stationary phases (underivatized silica, amide, diol, amino, zwitterionic, polysuccinimide, cyclodextrin, and mixed-mode) and optimization of HPLC conditions led to the identification of a Halo Penta HILIC column (4.6 × 150 mm, 5 µm) and a mobile phase comprising 85% acetonitrile and 15% ammonium formate buffer (100 mM, pH 3.2). Efficient separations of metoprolol, succinic acid, and EP metoprolol impurities M and N were achieved within a short time frame (<8 min). The HILIC-CAD method was subsequently validated per USP validation guidelines with respect to specificity, robustness, linearity, accuracy, and precision, and could be incorporated into the current USP-NF monographs to replace the outdated TLC methods. Furthermore, the developed method was successfully applied to determine organic impurities in metoprolol drug substance (metoprolol succinate) and drug products (metoprolol tartrate injection and metoprolol succinate extended release tablets).

Remotely Versus Locally Facilitated Simulation-based Training in Management of the Deteriorating Patient by Newly Graduated Health Professionals: A Controlled Trial.

INTRODUCTION: This study evaluated delivery of immersive simulation-based training (SBT) by distance education. Newly graduated health professionals' experience of and learning outcomes from videoconference-enabled remotely facilitated (RF) were prospectively compared with a locally facilitated (LF) format within a course addressing management of the deteriorating patient. METHODS: Participants were exposed to both RF and LF formats in an intervention course (IC). The primary outcome measure was a questionnaire detailing participants' experience of 1 RF scenario and 1 LF scenario. The 16-item questionnaire measured perceived learning, comfort, interaction with other learners and instructor, as well as quality of instruction, factors that are considered essential in both SBT and distance education. As a secondary outcome measure, learning outcomes, measured as precourse and postcourse scores and pass rates in multiple-choice question tests, were also measured and compared with those of participants completing control courses, in which only the LF format was used. RESULTS: The study was conducted between April 2013 and April 2014. Among the 155 participants who participated in ICs, questionnaire results revealed a small, significantly higher median total score (25-75 interquartile range) for LF versus RF format scenarios [78 (72-80) vs. 76 (68-80), P = 0.01]. Multiple-choice question test scores compared between 155 IC and 150 control course participants showed no significant differences. CONCLUSIONS: Participants' experience of SBT using the RF format was slightly less positive than the LF format; however, it had no measured impact on knowledge. The impact of RF-SBT on more complex training applications remains poorly understood. Instructors could potentially optimize learner comfort and engagement by improving their interactive skills.

Development and validation of a reversed-phase high-performance liquid chromatography method for routine identification and purity assessment of high-purity steviol glycoside sweeteners.

The widespread application of stevia-based sweeteners in food products has resulted in the need for reliable analytical methods for measuring the purity and identity of high-purity steviol glycoside ingredients. The objective of this research was to develop and validate a new reversed-phase separation method capable of separating and quantifying nine steviol glycosides present in typical high-purity stevia extract ingredients. Results of the study established the linearity of the method at a correlation factor of 1.000 for the two major components and other minor components of this food ingredient. Method accuracy values were in the range of 99.1-100.9%. The percent relative standard deviation for six independent assay determinations was 1.0%. The method was determined to be robust for minor changes in column temperature, initial acetonitrile content, flow rate, and wavelength. The validated high-performance liquid chromatography method was found to be suitable to be included by USP as a Food Chemicals Codex compendial standard for steviol glycosides.

The deep inferior epigastric perforator and pedicled transverse rectus abdominis myocutaneous flap in breast reconstruction: a comparative study.

BACKGROUND: Our objective was to compare the complication rates of two common breast reconstruction techniques performed at our hospital and the cost-effectiveness for each test group. METHODS: All patients who underwent deep inferior epigastric perforator (DIEP) flap and transverse rectus abdominis myocutaneous (TRAM) flap by the same surgeon were selected and matched according to age and mastectomy with or without axillary clearance. Patients from each resultant group were selected, with the patients matched chronologically. The remainder were matched for by co-morbidities. Sixteen patients who underwent immediate breast reconstruction with pedicled TRAM flaps and 16 patients with DIEP flaps from 1999 to 2006 were accrued. The average total hospitalisation cost, length of hospitalisation, and complications in the 2 year duration after surgery for each group were compared. RESULTS: Complications arising from both the pedicled TRAM flaps and DIEP flaps included fat necrosis (TRAM, 3/16; DIEP, 4/16) and other minor complications (TRAM, 3/16; DIEP, 1/16). The mean hospital stay was 7.13 days (range, 4 to 12 days) for the pedicled TRAM group and 7.56 (range, 5 to 10 days) for the DIEP group. Neither the difference in complication rates nor in hospital stay duration were statistically significant. The total hospitalisation cost for the DIEP group was significantly higher than that of the pedicled TRAM group (P<0.001). CONCLUSIONS: Based on our study, the pedicled TRAM flap remains a cost-effective technique in breast reconstruction when compared to the newer, more expensive and tedious DIEP flap.