A high-performance liquid chromatographic-atmospheric pressure chemical ionization-tandem mass spectrometric method for determination of risperidone and 9-hydroxyrisperidone in human plasma.

Risperidone, a benzisoxazole derivative, is an antipsychotic agent used for the treatment of schizophrenia. We developed a liquid chromatographic-atmospheric pressure chemical ionization-tandem mass spectrometric (LC-APCI-MS-MS) method with improved sensitivity, selectivity, and dynamic range for determination of risperidone and 9-hydroxyrisperidone in human plasma. A structural analogue of risperidone, RO68808 (5 ng/mL), is added as the internal standard to 1 mL of human plasma. Plasma is made basic, extracted with pentane/methylene chloride (3:1), the organic phase evaporated to dryness, and the residue is reconstituted in water with 0.1% formic acid/acetonitrile (20:1). For LC-MS-MS analysis, a Metachem Inertsel HPLC column (2.1 x 150 mm, 5-microm particle size) is connected to a Finnigan TSQ7000 tandem MS via the Finnigan API interface. Both electrospray (ESI) and APCI produced predominantly MH(+) ions for the two analytes and the internal standard. Ions detected by selected reaction monitoring correspond to the following transitions: m/z 411 to 191 for risperidone, m/z 427 to 207 for 9-hydroxyrisperidone, and m/z 421 to 201 for the internal standard. APCI provided a larger dynamic range (0.1 to 25 ng/mL) and better precision and accuracy than ESI. Intrarun accuracy and precision determined at 0.1, 0.25, 2.5, and 15 ng/mL were within 12% of target with %CVs not exceeding 10.9%. Interrun accuracy and precision determined at the same concentrations were within 9.6% of target with %CVs not exceeding 6.7%. Analytes were stable in plasma after 24 h at room temperature, 2 freeze-thaw cycles, and 490 days at -20 degrees C.

Opening doors and minds: a path for widening access.

BACKGROUND: Students from disadvantaged socio-economic backgrounds are under-represented in UK medical schools. Many successful interventions are also highly labour-intensive for medical schools to implement. We describe and evaluate a sustainable, low-cost strategy that provides participants with targeted support, advice and experience. METHODS: Year-12 participants (29-74 annually) from schools in areas of deprivation were paired with e-mentors from the medical student population. Engagement with this programme was used as one criterion to select approximately 20 mentees per year for participation in a 1-week summer school. All participants were offered consultant-led work experience during their summer holiday and were guaranteed places at a student-led outreach conference, where they received specific help with the writing of personal statements and interview skills. Summer school participants were followed-up by questionnaire to establish their career plans. RESULTS: We have delivered this programme annually for 3 years. All respondents to follow-up applied to study medicine, dentistry or a related bioscience, to degree level. The success rate of these disadvantaged students was similar to that of the general population of UK applicants who applied to study medicine at this medical school. DISCUSSION: By collaboratively linking multiple activities organised by the Outreach Office, academic staff and medical students, an annual cohort of approximately 20 participants from non-traditional backgrounds was provided with sustained support in preparing for applying to medical school. The limited data available from follow-up suggests that this approach may have helped overcome the social disadvantage facing these applicants.

Automating bioanalytical sample analysis through enhanced system integration.

BACKGROUND: In the bioanalytical laboratory, challenges associated with manual, repetitive, labor-intensive processes can be addressed by powerful automated liquid handlers; however, their implementation has been difficult due to lack of efficient integration into laboratory workflows. Faster throughput is afforded to ligand binding assay (LBA) technologies via enhanced automation, but the upstream sample processing still remains a bottleneck. To be truly efficient, these technologies must be incorporated into a laboratory information management system (LIMS) to streamline data analysis and reporting. RESULTS: Three off-the-shelf technologies that aid in improving bioanalytical laboratory efficiencies were utilized with minimal customization to streamline the sample analysis process. Information extracted via a sequence file from the LIMS run was utilized to perform the sample processing on the automated liquid handler. A file conversion tool converted the sequence files that allowed for sample processing and preparing the assay ready plate. The plate was then transferred to the LBA microfluidics platform to run the experiments. The integration was tested using a LBA PK assay that demonstrated good sample dilution and assay performance. CONCLUSION: We successfully integrated LIMS with an automated liquid handler and a microfluidics platform to automate the sample analysis process in the bioanalytical laboratory. The utilization of off-the-shelf technologies with minimal customization requires minimal resources from laboratory scientists. It may be possible to implement this approach for other analytical platforms.

Laboratory automation of high-quality and efficient ligand-binding assays for biotherapeutic drug development.

BACKGROUND: Regulated bioanalytical laboratories that run ligand-binding assays in support of biotherapeutics development face ever-increasing demand to support more projects with increased efficiency. Laboratory automation is a tool that has the potential to improve both quality and efficiency in a bioanalytical laboratory. The success of laboratory automation requires thoughtful evaluation of program needs and fit-for-purpose strategies, followed by pragmatic implementation plans and continuous user support. RESULTS: In this article, we present the development of fit-for-purpose automation of total walk-away and flexible modular modes. We shared the sustaining experience of vendor collaboration and team work to educate, promote and track the use of automation. CONCLUSION: The implementation of laboratory automation improves assay performance, data quality, process efficiency and method transfer to CRO in a regulated bioanalytical laboratory environment.

A liquid chromatographic-electrospray ionization-tandem mass spectrometric method for determination of buprenorphine, its metabolite, norbuprenorphine, and a coformulant, naloxone, that is suitable for in vivo and in vitro metabolism studies.

A liquid chromatographic-electrospray ionization-tandem mass spectrometric method has been developed and validated for determination of the antiabuse medication, buprenorphine, its primary metabolite, norbuprenorphine, and a proposed coformulant, naloxone. The method uses deuterated internal standards and a simple liquid-liquid extraction. Mass spectrometry employed selected reaction monitoring of the transitions of m/z 468 to 396 for buprenorphine, 472 to 400 for [2H4]buprenorphine, 414 to 101 for norbuprenorphine, 423 to 110 for [2H9]norbuprenorphine, 328 to 310 for naloxone, and 345 to 327 for its internal standard, [2H3]naltrexone. The method was accurate and precise across the dynamic range of 0.1 to 10 ng/ml. All analytes were stable in human plasma stored at room temperature for up to 24 h and after three freeze-thaw cycles. Reconstituted extracts were stable at -20 degrees C for up to 3 days. In human subjects receiving a sublingual tablet of 8 mg buprenorphine and 2 mg naloxone, buprenorphine and norbuprenorphine were detected for up to 24 h with respective maximum concentrations at 1 and 1.5 h. Maximal concentrations ranged from 2.2 to 2.8 and 1.5 to 2.4 ng/ml for buprenorphine and norbuprenorphine, respectively (i.e., approximately 6 nM). The method detected norbuprenorphine formation in human liver microsomes incubated with 5-82 nM buprenorphine, which encompasses the therapeutic plasma concentration range. When cDNA-expressed P450s were incubated with 21 nM buprenorphine, norbuprenorphine formation was detected for P450s 3A4, as previously described, but also for 3A5, 3A7, and 2C8. Buprenorphine utilization generally exceeded norbuprenorphine formation, suggesting that P450s 2C18, 2C19, 2D6, and 2E1 may also be involved in buprenorphine metabolism to other products. These results suggest this method is suitable for both in vivo and in vitro studies of buprenorphine metabolism to norbuprenorphine.