Development of a UHPLC-MS/MS (SRM) method for the quantitation of endogenous glucagon and dosed GLP-1 from human plasma.

AIM: The performance of glucagon and GLP-1 immunoassays is often poor, but few sensitive LC-MS/MS methods exist as alternatives. EXPERIMENTAL: A multiplexed LC-MS/MS method using a 2D extraction technique was developed. RESULTS: The method was established for the quantitation of endogenous glucagon (LLOQ: 15 pg/ml) and dosed GLP-1 (LLOQ: 25 pg/ml) in human plasma, and is the first such method avoiding immunoenrichment. Specificity of endogenous glucagon quantitation was assured using a novel approach with a supercharging mobile phase additive to access a sensitive qualifier SRM. Endogenous glucagon concentrations were within the expected range, and showed good reproducibility after extended sample storage. A cross-validation against established immunoassays using physiological study samples demonstrated some similarities between methods. CONCLUSION: The LC-MS/MS method offers a viable alternative to immunoassays for quantitation of endogenous glucagon, dosed glucagon and/or dosed GLP-1.

Identification of plasma protease derived metabolites of glucagon and their formation under typical laboratory sample handling conditions.

RATIONALE: Glucagon modulates glucose production, and it is also a biomarker for several pathologies. It is known to be unstable in human plasma, and consequently stabilisers are often added to samples, although these are not particularly effective. Despite this, there have not been any studies to identify in vitro plasma protease derived metabolites; such a study is described here. Knowledge of metabolism should allow the development of more effective sample stabilisation strategies. METHODS: Several novel metabolites resulting from the incubation of glucagon in human plasma were identified using high-resolution mass spectrometry with positive electrospray ionisation. Tandem mass spectrometric (MS/MS) scans were acquired for additional confirmation using a QTRAP. Separation was performed using reversed-phase ultra-high-performance liquid chromatography. The formation of these metabolites was investigated during a time-course experiment and under specific stress conditions representative of typical laboratory handling conditions. Clinical samples were also screened for metabolites. RESULTS: Glucagon(3-29) and [pGlu](3) glucagon(3-29) were the major metabolites detected, both of which were also present in clinical samples. We also identified two oxidised forms of [pGlu](3) glucagon(3-29) as well as glucagon(19-29), or 'miniglucagon', along with the novel metabolites glucagon(20-29) and glucagon(21-29). The relative levels of these metabolites varied throughout the time-course experiment, and under the application of the different sample handling conditions. Aprotinin stabilisation of samples had negligible effect on metabolite formation. CONCLUSIONS: Novel plasma protease metabolites of glucagon have been confirmed, and their formation characterised over a time-course experiment and under typical laboratory handling conditions. These metabolites could be monitored to assess the effectiveness of new sample stabilisation strategies, and further investigations into their formation could suggest specific enzyme inhibitors to use to increase sample stability. In addition the potential of the metabolites to affect immunochemistry-based assays as a result of cross-reactivity could be investigated.

Development of a high-throughput UHPLC-MS/MS (SRM) method for the quantitation of endogenous glucagon from human plasma.

BACKGROUND: Published LC-MS/MS methods are not sensitive enough to quantify endogenous levels of glucagon. RESULTS: An ultra high performance liquid chromatography-MS/MS (SRM) method for the quantitation of endogenous levels glucagon was successfully developed and qualified. A novel 2D extraction procedure was used to reduce matrix suppression, background noise and interferences. Glucagon levels in samples from healthy volunteers were found to agree with radioimmunoassay (RIA) derived literature values. Bland-Altman analysis showed a concentration-dependent positive bias of the LC/MS-MS assay versus an RIA. Both assays produced similar pharmacokinetic profiles, both of which were feasible considering the nature of the study. CONCLUSION: Our method is the first peer reviewed LC-MS/MS method for the quantitation of endogenous levels of glucagon, and offers a viable alternative to RIA-based approaches.

UHPLC for the separation of proteins and peptides.

There is increasing interest within the pharmaceutical industry in the development of proteins and peptides as drugs in addition to their use as biomarkers. Immunochemistry-based techniques have been traditionally used for the quantitation of proteins and peptides; however, LC-MS-based methodologies are being increasingly adopted as they offer several advantages. UHPLC is well established within the small-molecule community as a means to increase resolution and/or the speed of separations prior to MS detection; however, it is rarely applied to proteins or peptides separations. In this paper, current applications of UHPLC to such separations are reviewed, as well as considerations with regard to the effect of altering various chromatographic parameters.

Catalytic dioxygen activation by (nitro)(meso-tetrakis(2-n-methylpyridyl)porphyrinato)cobalt(III) cation derivatives electrostatically immobilized in nafion films: an experimental and DFT investigation.

Complexes of the (nitro)( meso-tetrakis(2- N-methylpyridyl)porphyinato)cobalt(III) cation, [LCoTMpyP(2)(NO 2)] (4+), in which L = water or ethanol have been immobilized through ionic attraction within Nafion films (Naf). These immobilized six-coordinate species, [LCoTMPyP(2)(NO 2)/Naf], have been found to catalyze the oxidation of triphenylphosphine in ethanol solution by dioxygen, therefore retaining the capacity to activate dioxygen catalytically without an additional reducing agent as was previously observed in nonaqueous solution for the non-ionic (nitro)cobalt porphyrin analogs. Heating these immobilized six-coordinate species under vacuum conditions results in the formation of the five-coordinate nitro derivatives, [CoTMPyP(2)(NO 2)/Naf] at 85 degrees C and [CoTMPyP(2)/Naf] at 110 degrees C. The catalytic oxidation of gas-phase cyclohexene with O 2 is supported only by the resulting immobilized five-coordinate nitro complex as was previously seen with the corresponding solution-phase catalyst in dichloromethane solution. The simultaneous catalytic oxidation of triphenylphosphine and cyclohexene with O 2 in the presence of the Nafion-bound six-coordinate ethanol nitro complex is also observed; however, this process is not seen for the CoTPP derivative in dichloromethane solution. The oxidation reactions do not occur with unmodified Nafion film or with Nafion-supported [BrCo(III)TmpyP]/Naf or [Co(II)TmpyP]/Naf, indicating the necessity for the nitro/nitrosyl ligand in the oxidation mechanism. The existence of a second reactive intermediate is indicated because the two simultaneous oxidation reactions depend on two distinct oxygen atom-transfer steps having different reactivity. The absence of homogeneous cyclohexene oxidation by the six-coordinate (H 2O)CoTPP(NO 2) derivatives in the presence of Ph 3P and O 2 in dichloromethane solution indicates that the second reactive intermediate is lost by an unidentified route only in solution, implying that the immobilization of it in Nafion allows it to react with cyclohexene. Although direct observation of this species has not been achieved, a comparitive DFT study of likely intermediates in several catalytic oxidation mechanisms at the BP 6-31G* level supports the possibility that this intermediate is a peroxynitro species on the basis of relative thermodynamic accessibility. The alternate intermediates evaluated include the reduced cobalt(II) porphyrin, the dioxygen adduct cobalt(III)-O 2 (-), the oxidized cobalt(II) pi-cation radical, and the nitrito complex, cobalt(III)-ONO.

Nutrient recovery from swine lagoon water by Spirodela punctata.

Spirodela punctata 7776, the best duckweed strain in total protein production selected from in vitro screening experiments with synthetic swine lagoon water medium was examined for N and P recovery. It has shown a capability to grow in and to remove N and P from synthetic swine lagoon water with high N (240 mg NH4 N/l) and P (31.0 mg PO4 P/l) levels. A lag period of approximately 96 h was observed before the duckweed started to grow. During the lag period, utilization of N and P by the duckweed was very slow. The rates of N and P uptake, and duckweed growth increased with the increase of the initial N and P concentrations in the medium. The highest rates of N and P uptakes, and duckweed growth observed in this study were 0.955. 0.129 mg/l-h, and 1.33 g/m2-h (or 31.92 g/m2-day), respectively. The N:P ratio in swine lagoon water is adequate for growing the duckweed.