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On-line preparation techniques for plasma and in vitro sampling

1st April 2013


The development of, and the need for, faster drug discovery techniques in a highly competitive pharmaceutical industry has resulted in ever increasing numbers of pre-clinical biological samples to be analysed. By Dr Denise Wailwroth.

High throughput screening and combinatorial chemistry has greatly expanded the pool of potential drug candidates. New in vitro techniques have provided the ability to reduce this list to some degree, but in vivo studies still make a major contribution to understanding the pharmacology, toxicology and efficacy of drugs in development. Obtaining adsorption, distribution, metabolism and elimination (ADME) parameters early on in the drug discovery process is important to reduce the failure rate of drug candidates in clinical trials. The result is a requirement for the speedy processing of both biological samples and microsomal incubations.

The extraction of biological samples prior to analysis is frequently time consuming. LC/MS/MS has become the detection method of choice for both speed and selectivity. Fast sample preparation techniques that keep pace with the speed of LC/MS/MS are now essential.

BioTrap and LC/MS/MS

Automated solid phase extraction or simple plasma protein precipitation both have their place and their disadvantages. On-line extraction is now available in various forms, including robotic SPE and 96 (or greater)-well plate systems, while on-line cartridge extraction systems have frequently been incompatible with LC/MS/MS analysis. Recent technology using the BioTrap particle enables on-line extraction with LC/MS/MS compatible solvents: moreover, generic screening methods have recently been developed that provide one standard extraction method for acids and one for bases, both of which are compatible with LC/MS/MS.

BioTrap cartridges utilise a particle that has a biocompatible external surface of a1-acid glycoprotein and a hydrophobic internal surface of C18 or C8. The pore size of the particle is chosen such that plasma proteins and other large particles are excluded, and are washed to waste, but drug molecules can enter and become adsorbed. The degree to which they are trapped depends on the internal chemistry chosen and the extraction mobile phase selected. The particle size of the BioTrap media is chosen to provide an extremely low back pressure under operating conditions. It is the outer surface of a1-acid glycoprotein ­ one of the most solvent compatible proteins available ­ that ensures that proteins do not adhere and cause increases in this back pressure. This results in a long cartridge life ­ often through several hundred ml of plasma. The advantages are: u The analytical method requires little or no alteration ­ a slight adjustment to the mobile phase may be necessary. u The choice of detection method need not change, although the more specific the detection method, the better the sensitivity of the method. u Analysis of total drug ­ not just free drug ­ is enabled, making it a sensitive technique even for highly protein bound compounds. u A long life and a high plasma injection volume (up to 1ml) makes this an economic automated extraction technique.

Using testosterone as a test analyte, the BioTrap on-line extraction technique has been investigated for its applicability to in vitro drug metabolism studies using Cytochrome P450 mediated metabolism. Hydroxylated metabolites and testosterone are usually extracted from microsomes by liquid-liquid extraction into methylene chloride or ethyl acetate. As an alternative, automated method, microsomal samples were directly injected onto the BioTrap C18 cartridge for on-line extraction and then switched onto the analytical column for analysis.

The drive towards a major reduction in animal usage for drug development has lead to new advances in the use of microdialysis for multiple sampling in a single animal. Microdialysis makes it possible to monitor relative changes in the chemical composition of living tissue without altering the fluid balance. In this way, sampling may be effected from blood brain barrier, muscle, bile duct, vein or skin simultaneously in a single animal. Information on the relative concentration of targeted analytes, in specific tissues of a living animal, can be obtained for periods from hours to a few days. Dialysates may be diverted to the loop of an injection valve on an LC, LC/MS or CE system for analysis. Because these tiny in vivo probes utilise a semi-permeable dialysis membrane, samples are already cleared of large molecular weight entities including proteins.

Direct blood sampling in small animals such as rats for longer periods of time (24 hrs) can be a real logistical challenge. A new fully automated system, Culex, is now completing development at BAS that enables all the ADME information required to be obtained from a single animal. Currently, regulatory and practical issues limit the numbers of blood samples that can be obtained from animals for drug development studies, affecting the quality of data obtained. In addition, the requirement for manual operation of this technique limits the number of animals that can be sampled simultaneously.

BAS research laboratories has prototyped a new instrument to better facilitate these processes. Automated sampling of systemic blood and brain dialysates is now possible in freely moving, cannulated animals. Refrigerated microfraction collection is used with a swivel-less animal containment system which also permits monitoring of both animal behaviour (degree and direction of movement) as well as metabolic factors (separate collection of urine and faeces). Blood sampling time points and volume are independently programmable through validated Windows software capable of a synchronised handling up to four animals over periods of 24 hours or more.

Depending on the analyte(s), pharmacokinetic data is obtained and metabolites may be identified with ion trap mass spectrometry. In brain dialysates, neurotransmitters may be monitored for pharmacodynamic information using LCEC with a four-channel electrochemical detector.





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