Quantification below the LLOQ in regulated LC-MS/MS assays: a review of bioanalytical considerations and cautions.

In response to an earlier workshop covering the pros and cons of quantification below the LLOQ (BLQ) the author reviews the topics discussed from the bioanalytical standpoint. Important considerations for estimating concentrations below the LLOQ include: method signal-to-noise, baseline shape and condition, close lying interference peaks (especially for protein methods), matrix effect, adsorption and stability of the analyte at low concentrations and carryover. These methodological issues are discussed as possible contributors to inaccuracy in BLQ estimations, and appropriate cautions are provided via examples. A proposed method for the evaluation of BLQ estimations utilizes extended incurred sample reanalysis analysis where BLQ samples or spiked simulated samples are analyzed with quality controls and standards in addition to those in the original study. Generally, BLQ estimations are discouraged, with the recommendation that any extrapolations should be done in close collaboration between the pharmacokinetic (PK) and bioanalytical scientists in consultation with the regulatory agency.

Liquid chromatographic-tandem mass spectrometric assay for the simultaneous quantification of Camptosar and its metabolite SN-38 in mouse plasma and tissues.

A simple, rapid and sensitive LC-MS/MS bioanalytical method has been developed to simultaneously quantify Camptosar (CPT-11) and its active metabolite, SN-38, in mouse plasma and tissues. A single step protein precipitation with acetonitrile in 96-well plates was used for sample preparation. Camptothecin (CPT) was used as the internal standard. Fast separation of SN-38, CPT-11 and CPT was carried out isocratically on a C18, 2 mm x 50 mm, 5 microm HPLC column with a mobile phase containing acetonitrile and 20 mM ammonium acetate (pH 3.5) and a 2.5 min chromatographic run time. The API 4000 MS/MS system was operated in positive ionization multiple reaction monitoring mode, and the transitions for SN-38, CPT-11 and CPT were 393.4 --> 349.3, 587.6 --> 167.2 and 349.3 --> 305.3, respectively. The SN-38 and CPT-11 concentrations in samples were calculated from a standard curve of peak area ratios of the analyte to that of the internal standard using a 1/chi2 weighted linear regression. The quantitation limit of 0.5 ng/mL was achieved by using a low sample volume (100 microL) of plasma or tissue homogenates. The assay was linear over the concentration range of 0.5-500 ng/mL with acceptable precision and accuracy. The method was used for the quantification of CPT-11 and SN-38 in plasma and tissues to support a preclinical pharmacokinetics and tissue distribution study of CPT-11 in mice.

Bioanalytical method validation considerations for LC-MS/MS assays of therapeutic proteins.

This paper highlights the recommendations of a group of industry scientists in validating regulated bioanalytical LC-MS/MS methods for protein therapeutics in a 2015 AAPSJ White Paper. This group recommends that most of the same precision and accuracy validation criteria used for ligand-binding assays (LBAs) be applied to LC-MS/MS-based assays where proteins are quantified using the LC-MS/MS signal from a surrogate peptide after proteolytic digestion (PrD-LCMS methods). PrD-LCMS methods are generally more complex than small molecule LC-MS/MS assays and may often include LBA procedures, leading to the recommendation for a combination of chromatographic and LBA validation strategies and appropriate acceptance criteria. Several key aspects of this bioanalytical approach that are discussed in the White Paper are treated here in additional detail. These topics include selectivity/specificity, matrix effect, digestion efficiency, stability and critical reagent considerations.

Recommendations for validation of LC-MS/MS bioanalytical methods for protein biotherapeutics.

This paper represents the consensus views of a cross-section of companies and organizations from the USA and Canada regarding the validation and application of liquid chromatography tandem mass spectrometry (LC-MS/MS) methods for bioanalysis of protein biotherapeutics in regulated studies. It was prepared under the auspices of the AAPS Bioanalytical Focus Group's Protein LC-MS Bioanalysis Subteam and is intended to serve as a guide to drive harmonization of best practices within the bioanalytical community and provide regulators with an overview of current industry thinking on applying LC-MS/MS technology for protein bioanalysis. For simplicity, the scope was limited to the most common current approach in which the protein is indirectly quantified using LC-MS/MS measurement of one or more of its surrogate peptide(s) produced by proteolytic digestion. Within this context, we considered a range of sample preparation approaches from simple in-matrix protein denaturation and digestion to complex procedures involving affinity capture enrichment. Consideration was given to the method validation experiments normally associated with traditional LC-MS/MS and ligand-binding assays. Our collective experience, thus far, is that LC-MS/MS methods for protein bioanalysis require different development and validation considerations than those used for small molecules. The method development and validation plans need to be tailored to the particular assay format being established, taking into account a number of important factors: the intended use of the assay, the test species or study population, the characteristics of the protein biotherapeutic and its similarity to endogenous proteins, potential interferences, as well as the nature, quality, and availability of reference and internal standard materials.

Inhibition of light modulation of chloroplast enzyme activity by sulfite : One of the lethal effects of SO2.

The capacity of a particulate pea (Pisum sativum L.) leaf chloroplast system for light-modulation of enzyme activity is diminished by brief exposure to sodium sulfite and, when intact seedlings are exposed to atmosphric SO2, the same system is inactivated. The destructive effect of this pollutant on green plants may therefore be due to disruption of the mechanism for control of carbon dioxide fixation.

Quantification of raf antisense oligonucleotide (rafAON) in biological matrices by LC-MS/MS to support pharmacokinetics of a liposome-entrapped rafAON formulation.

An LC-MS/MS method was developed to quantify an antisense oligonucleotide against Raf-1 expression (rafAON) in monkey and mouse plasma and in mouse tissue homogenates from animals dosed with a liposome-entrapped rafAON easy-to-use formulation (LErafAON-ETU) intended for use as an antineoplastic agent. RafAON was extracted from mouse and monkey plasma using solid-phase extraction. Tissues were homogenized and sample cleanup was achieved by protein precipitation. RafAON and the internal standard (IS) were separated on a Hamilton PRP-1 column and quantified by tandem mass spectrometry using an electrospray source in negative ion mode. The total run time was 4.0 min. The peak areas of two rafAON transitions were summed and plotted against the peak area of an IS transition to generate the standard curve. In monkey plasma the linear range was 50-10,000 ng/mL, and in mouse plasma it was 25-5000 ng/mL. The lower limit of quantification was 500 ng/mL (10 microg/g tissue) in heart, kidney, liver, lung and spleen homogenates, and the standard curve was linear up to 10,000 ng/mL. Accuracy, precision and stability were evaluated and found to be acceptable in all three matrices. The assay was used to support pharmacokinetics and tissue distribution studies of LErafAON-ETU in mice and monkeys.