BA Method Development: Blood Specimen – BioPharma Services

The development of new drugs is a long, complex, expensive, and multidisciplinary process. Drug discovery and development takes more than 10 years, and the average cost of each new drug approved for clinical use is more than $1 billion. In addition, 90% of drug development projects fail during the clinical development phase. Du Xin Sun et al summarized the failure rates at each stage of drug development. In Fig 1, they classified drug candidates into four different categories based on potency/selectivity, tissue exposure/selectivity, and dose required to balance clinical efficacy/toxicity. Class I, excellent efficacy, and low toxicity. Class II, adequate efficacy, and high toxicity. Class III, adequate efficacy, and controlled toxicity. Class IV, low efficacy, and high toxicity.

Among them, Class III drugs can achieve good efficacy at small or moderate doses, have high selectivity for diseased organs and low selectivity for normal organs, and have low concentrations in human plasma. Due to the low plasma exposure of the drug, such drugs are often terminated at an early stage of the drug optimization process.

As a result, pharmacokinetic analyses using plasma data can sometimes be misleading, whereas pharmacokinetic analyses using data from whole blood, or other biological samples are sometimes more reliable. Considerations for matrix selection in LC-MS bioanalysis become critical in the toxicokinetic and pharmacokinetic assessment of drug development, especially in bioanalytical method development for blood specimens.

Fig. 1. The process of drug discovery and development, and the failure rate at each step.

Why there is a need to develop bioanalytical methods for blood samples.

Blood is a very important fluid in the body: it is thicker than water, feels a little sticky, and carries oxygen from the lungs to the body’s cells for metabolic needs. Whole blood contains mainly red blood cells (or erythrocytes), white blood cells (or leukocytes), and platelets (or thrombocytes) suspended in plasma (the liquid part of blood). Generally, drugs are transported in the circulation in both free (unbound) and bound (bound to plasma proteins and red blood cells) forms. Certain compounds are preferentially sequestered into red blood cells rather than plasma.

The extent to which a drug binds to plasma proteins or erythrocytes depends on its physicochemical properties, binding site, and binding capacity. Biological fluids such as plasma, serum, whole blood, and urine are commonly analyzed for pharmacokinetic parameter assessment during drug discovery and development. Most bioanalytical applications come from plasma samples.

However, for many compounds such as cyclosporin A (CsA), which is predominantly distributed in erythrocytes, whole blood, rather than plasma or serum, is the preferred matrix for the measurement of drug exposure in animals or humans. This highlights the importance of bioanalytical method development in blood specimens.

What are the challenges to developing bioanalytical methods in blood specimens?

Whole blood and all four of its components are important tools in modern medicine. These matrix components in blood samples affect the response of the analytes of interest and can also lead to inaccurate quantification, and therefore must be addressed in bioanalytical method development of blood specimen.

Compared to plasma samples, whole blood samples pose unique challenges for bioanalytical method development and validation due to their viscosity and compositional complexity. Sample preparation techniques for whole blood assays are as diverse as plasma assays.

Although simple sample preparation techniques such as protein precipitation (PPT) have been used for whole blood analysis, whole blood analysis usually requires labor-intensive processing such as liquid-liquid extraction (LLE) or solid phase extraction (SPE). In some cases, PPT is used as a pretreatment method prior to liquid-liquid extraction or solid-phase extraction.

Case Study: Determination of Hydroxychloroquine in human blood by LC-MS/MS

Hydroxychloroquine is the standard of care in the treatment of systemic lupus erythematosus, rheumatoid arthritis (RA), and other inflammatory rheumatic diseases. For application in a bioequivalence study, a bioanalytical method to analyze hydroxychloroquine in human blood has been successfully developed at BioPharma Services. The linear dynamic range of the method is from 2.00 to 400.00 ng/mL.

The assay utilizes a supported liquid extraction procedure requiring 50 µL of human whole blood spiked with stable label internal standard (Hydroxychloroquine-d4). Samples are chromatographically separated on a Phenomenex Luna C18 analytical column. The AB SCIEX QTRAP 5500 mass spectrometric instrument is operated in positive ion mode using a Turbo IonSpray source and MRM detection. The total run time for a 10.0 µL injection is 5.0 minutes per sample. Three metabolites of hydroxychloroquine were tested for interference during the validation: Desethyl Chloroquine (100.00 ng/mL in whole blood), and Didesethyl Chloroquine (100.00 ng/mL in whole blood) Desethylhydroxychloroquine (Cletoquine) (500.00 ng/mL in whole blood).

There is no impact on the quantitation of Hydroxyleucine. Some chromatograms show great sensitivity of the method (Fig.2) and good separation of hydroxychloroquine and its metabolites. (Fig.3) The method was fully validated and apply to two pilot biostudies without fail batches.

Fig2. Chromatogram for whole blood blank and LLOQ – whole blood bank.

Fig2.1 Chromatogram for whole blood blank and LLOQ-LLOQ (2.00 ng/mL) in Whole blood.

Fig 3. The Separation of hydroxychloroquine and its metabolites.

Why Choose BioPharma Services for your next Drug Development Project.

During drug development, plasma rather than whole blood has been the primary biological matrix for toxicokinetic (TK) or pharmacokinetic (PK) evaluation of drug candidates. However, for certain drugs that bind or sequester to red blood cells, whole blood is more suitable than plasma for toxicokinetic (TK) or pharmacokinetic (PK) evaluation. Whole blood samples are typically more difficult to process and assay than serum or plasma samples. Currently, there are no specific guidelines for the selection of biological substrates for TK/PK assessment of drugs. Therefore, it is imperative to understand the implications associated with substrate selection and how it can be facilitated in bioanalysis.

At BioPharma, our knowledgeable and experienced bioanalysis team develops accurate and efficient analytical methods according to customer requirements. All methods are validated in accordance with FDA, EMA, and ICH M10 guidelines to ensure the accuracy, reliability, and integrity of all data.

Written by: Hongzhi Liu, Principal Research Scientist.