A validated LC-MS/MS method for the quantitative measurement of creatinine as an endogenous biomarker in human plasma.

BACKGROUND: Creatinine is an endogenous compound generated from creatine by normal muscular metabolism. It is an important indicator of renal function and the serum level is routinely monitored in clinical labs. Results & methodology: Surrogate analyte (d3-creatinine) was used for calibration standard and quality control preparation and the relative instrument response ratio between creatinine and d3-creatinine was used to calculate the endogenous creatinine concentrations. CONCLUSION: A fit-for-purpose strategy of using a surrogate analyte and authentic matrix was adopted for this validation. The assay was the first human plasma assay using such strategy and was successfully applied to a clinical study to confirm a transient elevation of creatinine observed using an existing clinical assay.

Bioanalysis of dried saliva spot (DSS) samples using detergent-assisted sample extraction with UHPLC-MS/MS detection.

Dried saliva spot (DSS) sampling is a non-invasive sample collection technique for bioanalysis that can be potentially implemented at the patient's home. A UHPLC-MS/MS assay was developed using detergent-assisted sample extraction to quantify BMS-927711, a drug candidate in development for the treatment of migraines, in human DSS. By implementing DSS sampling at the patients' home, the bioanalytical sample collection for pharmacokinetic evaluation can be done at the time of the acute migraine attack without the need for clinical visits. DSS samples were prepared by spotting 15 µL of liquid saliva onto regular Whatman FTA™ DMPK-C cards and verified with a UV lamp (at λ 254 nm or 365 nm) during DSS punching. The 4-mm DSS punches in a 96-well plate were sonicated with 200 µL of [(13)C2, D4]-BMS-927711 internal standard (IS) solution in 20/80 MeOH/water for 10 min, followed by sonication with 50 µL of 100 mM NH4OAc with 1.0% Triton-X-100 (as detergent) prior to liquid-liquid extraction with 600 µL EtOAc/Hexane (90:10). UHPLC-MS/MS was performed with an Aquity(®) UPLC BEH C18 Column (2.1 × 50 mm, 1.7 µm) on a Triple Quad™ 5500 mass spectrometer. The assay was linear with a concentration range from 2.00 to 1000 ng mL(-1) for BMS-927711 in human saliva. The intra- and inter-assay precision was within 8.8% CV, and the accuracy was within ±6.7% Dev of the nominal concentration values. This UHPLC-MS/MS assay has been successfully applied to determine the drug's pharmacokinetics within a clinical study. For the first time, we observed BMS-927711 exposure in human DSS, confirming the suitability of this sampling technique for migraine patients to use at home. Detergent-assisted extraction with Triton-X-100 could be very useful in DSS or other dried matrix spot (DMS) assays to overcome low or inconsistent analyte recovery issues.

Development and validation of a liquid chromatography tandem mass spectrometry assay for the quantitation of a protein therapeutic in cynomolgus monkey serum.

We have developed and fully validated a fast and simple LC-MS/MS assay to quantitate a therapeutic protein BMS-A in cynomolgus monkey serum. Prior to trypsin digestion, a recently reported sample pretreatment method was applied to remove more than 95% of the total serum albumin and denature the proteins in the serum sample. The pretreatment procedure simplified the biological sample prior to digestion, improved digestion efficiency and reproducibility, and did not require reduction and alkylation. The denatured proteins were then digested with trypsin at 60 °C for 30 min and the tryptic peptides were chromatographically separated on an Acquity CSH column (2.1 mm × 50 mm, 1.7 µm) using gradient elution. One surrogate peptide was used for quantitation and another surrogate peptide was selected for confirmation. Two corresponding stable isotope labeled peptides were used to compensate variations during LC-MS detection. The linear analytical range of the assay was 0.50-500 µg/mL. The accuracy (%Dev) was within ± 5.4% and the total assay variation (%CV) was less than 12.0% for sample analysis. The validated method demonstrated good accuracy and precision and the application of the innovative albumin removal sample pretreatment method improved both assay sensitivity and robustness. The assay has been applied to a cynomolgus monkey toxicology study and the serum sample concentration data were in good agreement with data generated using a quantitative ligand-binding assay (LBA). The use of a confirmatory peptide, in addition to the quantitation peptide, ensured the integrity of the drug concentrations measured by the method.

A novel and cost effective method of removing excess albumin from plasma/serum samples and its impacts on LC-MS/MS bioanalysis of therapeutic proteins.

We have developed an innovative method to remove albumin from plasma/serum samples for the LC-MS/MS quantitation of therapeutic proteins. Different combinations of organic solvents and acids were screened for their ability to remove albumin from plasma and serum samples. Removal efficiency was monitored by two signature peptides (QTALVELVK and LVNEVTEFAK) from albumin. Isopropanol with 1.0% trichloroacetic acid was found to be the most effective combination to remove albumin while retaining the protein of interest. Our approach was compared with a commercial albumin depletion kit on both efficiency of albumin removal and recovery of target proteins. We have demonstrated that our approach can remove 95% of the total albumin in human plasma samples while retaining close to 100% for two of three therapeutic proteins tested, with the third one at 60-80%. The commercial kit removed 98% of albumin but suffered at least 50% recovery loss for all therapeutic proteins when compared to our approach. Using BMS-C as a probe compound, the incorporation of the albumin removal approach has improved both assay sensitivity and ruggedness, compared to the whole plasma protein digestion approach alone. An LC-MS/MS method was developed and validated based on this new approach for the analysis of BMS-C in monkey serum. This assay was successfully applied to a toxicological study. When the albumin removal method was used in another clinical LC-MS/MS method, the sensitivity improved 10-fold to 50 ng/mL LLOQ comparing to a typical pellet digestion method.

Improved liquid-liquid extraction with inter-well volume replacement dilution workflow and its application to quantify BMS-927711 in rat dried blood spots by UHPLC-MS/MS.

An UHPLC-MS/MS method was developed and validated to quantify BMS-927711, a drug candidate to treat migraine, in rat dried blood spots (DBS). The DBS samples were extracted using an improved liquid-liquid extraction (LLE) strategy involving in the sonication of DBS punches in 20% MeOH aqueous solution containing the internal standard, [(13)C2, D4]-BMS-927711, and then with a 100mM NH4OAc buffer solution, followed by an automated LLE with EtOAc-hexane (70:30, v/v). The presence of 20% MeOH as an organic modifier in the elution solution significantly improved the analyte elution efficiency and assay performance. A novel inter-well volume replacement dilution workflow was introduced for DBS sample dilution before LLE step. This was a simple two-step process, firstly a small portion of the DBS blank solution was discarded, and then the same volume of a concentrated DBS sample solution was spiked into the leftover blank solution to achieve a desired dilution. Chromatographic separation was achieved on an Acuity UPLC(®) BEH C18 column (2.1mm×50mm, 1.7µm) and the analyte was detected by selected reaction monitoring (SRM) with positive electrospray ionization on an AB Sciex Triple Quad 5500 mass spectrometer. The standard curve was linear from 5.00 to 5000ng/mL with assay precision ≤4.9% CV, and assay accuracy within ±3.1%Dev of the nominal values. Accurate sample dilution was achieved by using inter-well volume replacement with a precision of ≤4.2% CV and an accuracy of ±3.3% for dilution QC at 50,000ng/mL with 100-fold dilution (n=18). This robust UHPLC-MS/MS assay has been successfully applied to the non-clinical studies in rats. By using inter-well volume replacement workflow, accurate dilution was demonstrated using only one DBS blank sample for a typical dilution of <50-fold, and using only two blank DBS samples for a dilution of up to 625-fold. Moreover, this new workflow makes it easier to automate DBS sample dilution.