Guowen Liu

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.

RATIONALE: Nucleotide analogs are highly polar and ionic, which impose great challenges on bioanalysis. Ion‐pairing liquid chromatography/tandem mass spectrometry (LC/MS/MS) is the predominant reported approach for such compounds. Assay ruggedness of ion‐pairing LC/MS/MS methods was often a challenge due to the potential contamination of the ion source of the mass spectrometer and LC column performance deterioration caused by ion‐pairing reagents. METHODS: An ion‐pairing reagent was only added to the reconstitution solution to minimize its exposure to the MS ion source. To achieve optimum sensitivity, high pH mobile phases and negative ion ESI were needed for the LC/MS/MS method. However, high pH mobile phases led to the accumulation of ion‐pairing reagent on the analytical column, which was washed off with an acidic solution to restore the column performance. In addition, isopropanol was used as a mobile phase modifier to improve peak shape and sensitivity. RESULTS: The limit of detection was established at 1.0 ng/mL in the cell lysate. The calibration curve showed good linearity over the range of 1.0 to 100 ng/mL. The overall accuracy was no less than 87.7% based on four levels of quality control samples. Inter‐run precision and intra‐run precision across four analytical runs for low, geometric, medium and high QCs were less than 12.9. CONCLUSIONS: By identifying and addressing the root cause of the assay ruggedness problem, we have developed a rugged ion‐pairing LC/MS/MS method for a triphosphate (TP) metabolite of BMS‐986001 in peripheral blood mononuclear cells. The new method overcame challenges such as a rapid deterioration of the peak shape, increased carryover and extremely poor column life. The peak shape was well maintained throughout multiple analytical runs. This method has been successfully applied to a toxicology study in cynomolgus monkey. Copyright © 2012 John Wiley & Sons, Ltd.

Unstable drug candidates often lead to complexity for both sample collection and bioanalysis. Dried blood spot (DBS) technology is believed to be a viable solution to address this problem. However, it is currently a challenge to evaluate compound stability on DBS due to its solid format. The observed compound loss on a DBS card could be degradation and/or incomplete recovery. Therefore, a reliable bioanalytical method which can differentiate recovery loss from degradation is necessary for such stability evaluation. In this paper, the stability of an unstable drug candidate (KAI-9803) in human blood was evaluated using DBS. A reliable approach to evaluating analyte stability on DBS was developed with an appropriate time-zero sample, a consistent DBS sample processing method, and a suitable positive control. Commercially available DBS cards were evaluated, and it was found that KAI-9803 degraded during the drying process. An in-house modified DBS card was developed and demonstrated to be able to stabilize KAI-9803 during the drying process by rapidly lowering the pH of the spotted blood sample. The storage stability of KAI-9803 in human blood on this new card has been established for at least 48 days at room temperature. This in-house modified DBS card could provide a generic approach for other compounds which require stabilization at a low pH.

Dried blood spot (DBS) technology is an emerging alternative for sample collection in bioanalysis. Dilution for DBS samples is a challenge due to its solid sample format. Currently, DBS samples requiring dilution were first extracted as regular samples and then diluted with extracted blank samples containing internal standard (IS). Since the dilution step is a volume‐critical step, extra care has to be taken to achieve accurate dilution when dealing with limited volume extracted samples. Here, we introduce an alternative sample dilution for liquid chromatography/tandem mass spectrometry (LC/MS/MS) assays using IS to track the dilution step. Dilution factor‐adjusted IS working solution was first added to the sample requiring dilution before sample processing; subsequently, the processed sample was approximately diluted into the assay linear response range before LC/MS/MS analysis. We define this approach as “IS‐tracked dilution”. The advantage of this approach is that the diluting step is tracked by the IS and is no longer a volume‐critical step. Another recognized challenge related to sample dilution is automatic sample dilution using a liquid handler. This “IS‐tracked dilution” may also help address some of the challenges for automatic sample dilution of liquid samples. This new dilution approach was proven to be effective and convenient in both plasma assays and DBS assays using omeprazole as a probe compound. Copyright © 2011 John Wiley & Sons, Ltd.

Matrix ion suppression/enhancement is a well observed and discussed phenomenon in electrospray ionization mass spectrometry Nonuniform matrix ion suppression/ enhancement across different types of samples in an analytical run is widely believed to be well compensated for by using a stable isotope labeled internal standard (SIL-IS) in bioanalysis using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) Therefore, the risk of nonuniform matrix ion suppression/ enhancement is usually deemed low when an SIL-IS is used Here, we have identified, evaluated, and proposed solutions to control bioanalytical risks from nonuniform matrix ion suppression/enhancement even with an SIL-IS through a case study using omeprazole Two lots of human blank urine were tested, and ion enhancement of about 500% for omeprazole was observed in one lot but not in the other When a quadratic regression model had to be used, the assay failed the industry acceptance criteria due to unacceptable positive bias for the middle and high quality control (QC) samples The failure was attributed to different extents of matrix ion enhancement between the standards (SIDs) and QCs, which resulted in the misaligned results from the regression model It was concluded that, for the same amount of drug, non uniform ion enhancement for different types of samples (STD or QC) resulted in different ion intensities, therefore leading to different response behaviors (linear or nonlinear) at the mass spectrometer detector A simplified mathematical model was used to evaluate the risk when unmatched response models occurred for different types of samples A diagnostic factor Q (Q = X-uLoQ (-A/B)) was proposed to monitor the risks, where X-uLOQ is the upper limit of quantitation of the assay, A is the quadratic slope of the curve, and B is the linear slope of the curve The potential maximum errors were estimated on the basis of the mathematical model for different scenarios, and Q values were given to control the risks under these conditions for bioanalysis using LC - MS/MS