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A simple procedure for selecting the correct weighting factors for linear and quadratic calibration curves with least-squares regression algorithm in bioanalytical LC-MS/MS assays is reported. The correct weighting factor is determined by the relationship between the standard deviation of instrument responses (σ) and the concentrations (x). The weighting factor of 1, 1/x, or 1/x2 should be selected if, over the entire concentration range, σ is a constant, σ2 is proportional to x, or σ is proportional to x1 respectively. For the first time, we demonstrated with detailed scientific reasoning, solid historical data, and convincing justification that 1/ x2 should always be used as the weighting factor for all bioanalytical LC-MS/MS assays. The impacts of using incorrect weighting factors on curve stability, data quality, and assay performance were thoroughly investigated. It was found that the most stable curve could be obtained when the correct weighting factor was used, whereas other curves using incorrect weighting factors were unstable. It was also found that there was a very insignificant impact on the concentrations reported with calibration curves using incorrect weighting factors as the concentrations were always reported with the passing curves which actually overlapped with or were very close to the curves using the correct weighting factor. However, the use of incorrect weighting factors did impact the assay performance significantly. Finally, the difference between the weighting factors of 1/x2 and 1/y2 was discussed. All of the findings can be generalized and applied into other quantitative analysis techniques using calibration curves with weighted least-squares regression algorithm. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 SILlS through a case study using omeprazole. Two Iota 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 (STDs) and QCs, which resulted in the misaligned results from the regression model. It was concluded that, for the same amount of drug, nonuniform 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 = XULOQ(-A/B)) was proposed to monitor the risks, where XULOQ is the upper limit of quantitalion 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 [ABSTRACT FROM AUTHOR]

Here we report a strategr for rapid method development of high-throughput bioanalytical assays using ultra high-performance liquid chromatography coupled with tandem mass spectrometry (uHPLC—MS/MS). First, a data set was established for the removal of representative phospholipids under different sample treatments to guide subsequent method development for various compounds. The recovery information of the analyte(s) of interest under different extraction conditions was then obtained during method development. With the recovery proffles and the pre-established knowledge of phospholipids removal in place, an optimal extraction condition was identified to give not only a satisfactory recovery but also a good cleanup of the sample. A rapid LC or uHPLC method was developed without the need of extensive column wash after the elution of the analyte. This strategy was demonstrated through the method development of a uHPLC—MS/MS bioanalytical assay for the quantitation of ketoconazole in human plasma with liquid—liquid extraction using a hexane and ethyl acetate solvent system. The retention time for ketoconazole through an isocradc elution was 18 s. Good accuracy and precision were obtained. Assay ruggedness was demonstrated by consistent internal standard responses and retention time for 500 sequential injections. In addition, consistent results were obtained for incurred sample reanalysis. [ABSTRACT FROM AUTHOR]