Simultaneous extraction and analysis of multiple cystine-dense peptides by µSPE and microflow-MS/MS from plasma.

Aim: Develop a universal extraction and liquid chromatography-mass spectrometer method to simultaneously analyze cystine-dense peptide (CDP) miniproteins from rat and human plasma. The results of the analysis will be used to assist selection of therapeutic drug candidates from the vast CDP library. Methods & results: A micro-elution solid-phase extraction method was developed for the sample preparation of the CDP peptides in rat and human plasma followed by analysis by microflow liquid chromatography MS/MS. The methods developed for drug discovery were found to be accurate (±≤15.2% from nominal concentrations) and precise (≤13.4% CV), with a dynamic range of 1.00-500 ng/ml and extraction recoveries of 47.2-99.0%. Conclusion: This bioanalytical method can be utilized to screen CDP proteins and other miniproteins for drug discovery, candidate selection and further drug development.

Recent advances in the bioanalytical applications of dried matrix spotting for the analysis of drugs and their metabolites.

DBS techniques for the bioanalysis of drugs and metabolites from whole blood have been demonstrated to be a useful tool in drug development. The term dried matrix spot (DMS) has been used to indicate that the DBS technique has been applied to nonblood matrices. DMS methods often employ a color-indicating process that enhances the ability to analyze these mostly transparent fluids when spotted onto collection paper. The color-indicating dye allows the analyst to visually confirm the location of the dried sample spot. Other benefits of using a color-indicating dye include improved method accuracy and precision, because the process of adding the dye allows for the concurrent addition of the IS prior to sample addition and extraction. To date, matrices that have been analyzed using DMS include cerebrospinal fluid, synovial fluid, saliva, tears, urine and plasma.

The advantages of microflow LC-MS/MS compared with conventional HPLC-MS/MS for the analysis of methotrexate from human plasma.

BACKGROUND: In support of bioanalysis, there has always been a desire to improve detection limits and reduce scale. Microflow LC (MFLC) coupled with MS accomplishes both of these goals. RESULTS: As such, MFLC coupled with an MS system was used to generate bioanalytical validation data that met US FDA criteria. The MFLC-MS/MS data was compared with the same method with the use of conventional HPLC-MS/MS and a more than 14× S/N improvement was found with the MFLC-MS/MS method. Methotrexate was used as a model molecule to demonstrate the validation of the method from human plasma. The MFLC-MS/MS method was demonstrated to be accurate (±7%) and precise (12.9% at the LLOQ and a maximum of 11.6% at all other concentrations) across the dynamic range of the assay (1-1000 ng/ml) and compared well with the HPLC-MS/MS method. The MFLC bioanalytical validation was performed at a flow rate of 35 µl/min on a 0.5-mm inner diameter (I.D.) column, whereas, for the same linear velocities on the 2.0-mm I.D. column, the conventional HPLC bioanalytical validation was performed at 700 µl/min. Since the flow rate of the MFLC system is 20-times less than the HPLC system, the consumable solvent and disposal cost to perform the MFLC validation was significantly less. CONCLUSION: MFLC-MS/MS can be used to perform bioanalytical method validations with increased MS signal, reduced source contamination and reduced solvent consumption.

Maternal plasma advanced glycation end products concentrations in response to oral 50-gram glucose load in mid-pregnancy: a pilot study.

BACKGROUND: Accumulating evidence documents the initiation of diverse physiologic and biochemical responses subsequent to an oral glucose load. OBJECTIVES: We sought to evaluate the extent to which acute hyperglycemia, resulting from a 50-gram glucose load, contributes to changes in maternal plasma concentrations of advanced glycation end products (AGEs), a heterogeneous group of molecules formed from the non-enzymatic reaction of reducing sugars with free amino groups of proteins, lipids, and nucleic acids. METHODS: Blood specimens were collected from each participant in mid-pregnancy using standard procedures before and after a 50-gram oral glucose load. Maternal plasma methylglyoxal (MG), pentosidine and N(epsilon)-(carboxymethyl)lysine (CML) (free and bound) were measured by HPLC-MS/MS method. Non-parametric methods were employed for statistical analysis. RESULTS AND CONCLUSIONS: Median plasma MG increased 1.27 fold as a result of acute hyperglycemia. Median bound CML concentrations were elevated 21% in post-load plasma samples as compared with pre-load samples, while median free pentosidine concentrations were 51% lower (both p-values < 0.05). Future studies of larger populations and longer periods of follow-up are warranted to investigate the consequences of acute and chronic hyperglycemia on placental function and fetal development.

Comparison of oral and transdermal administration of rasagiline mesylate on human melanoma tumor growth in vivo.

BACKGROUND/AIMS: Transdermal patch administration results in a locally high concentration of drug that induce local toxicity, including tumorogenicity. As a worst-case scenario for consequences of repeated application on neoplastic growth, the melanin-binding drug, rasagiline, was used in a transdermal formulation applied directly to a human-derived melanoma to determine the effects on tumor growth. MATERIALS AND METHODS: Rasagiline mesylate was administered either orally or transdermally to athymic mice implanted with human melanoma (SKMEL28) to determine the effects on tumor growth and survival. Over a 21-day period, animals were administered daily oral gavage (15 mg/kg) or one or two rasagiline mesylate transdermal patches every 3 days. After the last dose administration, blood samples were collected to confirm drug exposure. RESULTS: All animals from the untreated, vehicle and rasagiline groups survived to the end of the study; however, 7 out of the 10 cisplatin-treated animals died before the end of the study. Rasagiline mesylate dosed either via the oral or transdermal routes had comparable plasma exposure and, unexpectedly, significantly reduced absolute tumor volumes and tumor growth rates in the nude mouse SKMEL28 xenograft model. CONCLUSION: Transdermal delivery of melanin-binding rasagiline does not increase melanoma growth in the xenograft model. Because rasagiline decreases melanoma growth, it may be candidate for combination therapy for melanoma.

Use of conventional bioanalytical devices to automate DBS extractions in liquid-handling dispensing tips.

BACKGROUND: Conventional liquid-handling devices were employed, along with an improved punching device, to semi-automate dried blood spot (DBS) extraction of alprazolam, α-hydroxyalprazolam and midazolam from human whole blood. Liquid-handling devices were used to add internal standard to the DBS cards and to extract the analytes from the DBS, in order to be analyzed by HPLC-MS/MS. RESULTS: The technique was shown to be accurate (±12.0%) and precise (10.3%) across the dynamic range of the assay. CONCLUSION: The semi-automated extraction reduced sample preparation time by more than 50% when compared with more conventional DBS manual extraction methods.

Validation of HPLC-MS/MS methods for analysis of loxapine, amoxapine, 7-OH-loxapine, 8-OH-loxapine and loxapine N-oxide in human plasma.

BACKGROUND: Two ESI-LC-MS/MS methods were validated for the quantitative analysis of loxapine, amoxapine, 7-OH-loxapine, 8-OH-loxapine and loxapine N-oxide in human K(2)EDTA plasma. Cation-exchange solid-phase extraction (SPE) was used to extract loxapine, amoxapine and the two hydroxylated metabolites, and organic precipitation was used to quantify loxapine N-oxide. RESULTS: Both methods were shown to be accurate (±13%), intra-assay precision was less than 15%, and inter-assay precision was less than 10% in all instances across the entire dynamic range of the assays (0.0500-50.0 ng/ml for the SPE method and 0.100-25.0 ng/ml for the precipitation method). CONCLUSION: The validated methods for loxapine, amoxapine, 7-OH-loxapine, 8-OH-loxapine and loxapine N-oxide have been used to successfully support clinical trials.

Development and validation of an HPLC-MS/MS method for the analysis of dexamethasone from pig synovial fluid using dried matrix spotting.

BACKGROUND: Dried matrix spot techniques were employed to validate an HPLC-MS/MS assay for the determination of dexamethasone in clear Yorkshire pig synovial fluid using 15 µl of sample. We have adopted the term dried matrix spot to indicate that the techniques used for dried blood spots can be applied to nonblood matrices. The dried matrix spot method employs a color-indicating process developed at Alturas Analytics that enhances the ability to analyze transparent fluids spotted onto collection paper by allowing the analyst to visually verify the location of the dried sample spot. RESULTS: The method was shown to be accurate (±4.3%) and precise (14.2% at the LLOQ and ≤10.0% at all other concentrations) across the dynamic range of the assay. CONCLUSION: The method shows the potential application of dried matrix spot techniques for the analysis of transparent biological fluids.