Plasma sampling protocol

In recent years pharmaceutical scientists have participated in lively discussions about how present methods of bioequivalency determination might legitimately and advantageously be modified. For example, the question of whether it is necessary to always take plasma samples so that AUC at the end of the test is at least 90% of AUC at time infinity has been explored [7,8], Statistical aspects of this and other possible methods of modifying protocol design are covered in a most useful book published in 1999 and in several papers [8-10,22],... 

A standard double-sandwich enzyme-linked immunosorbent assay (ELISA) or Western blot analysis is used. As the concentration of factor is very low in normal plasma (approximately 0.1 mg/1 for factor VIII), it is necessary to subject plasma samples to cryoprecipitation in order to concentrate the sample, prior to Western blot analysis. The cryoprecipitation protocol described by Bi et al. is as follows (Bi et al., 1996 Sarkar et al., 2000 Mah et al., 2003). Plasma samples are collected as described above for the Coatest assay. Plasma is then frozen at -80 °C overnight. Frozen samples are then subject to centrifugation at 7000 x g for 20 min at 4°C. The precipitate is washed with... 

FIGURE 8.4 (A) A compartmentai model of drug behavior in the body. (B) An experinaental protocol on (A), showrjng drug administration (hold arrow) and plasma sampling (dashed line with bullet). 

For quantitation of HIV-1 virus load in serum/plasma samples, testing samples in duplicate is recommended. Samples are considered positive and are quantifiable when duplicate test results are positive. Samples with negative or discordant duplicate results are considered Amp-RT negative. Quantitative detection of Amp-RT PCR amplicons is made by an ELISA-based, nonradio-active oligoprobing system. The system uses streptavidin-coated microtiter wells that capture the biotin-labeled PCR product (DIG-detection ELISA). The protocol for the ELISA is as follows ... 

For this reason, when a cassette is composed of structurally diverse compounds, it is usually prudent to use protein precipitation as a plasma sample preparation technique. Although the absolute recovery will vary from compound to compound, this approach can be applied with fairly universal success across a wide structural range of compounds and assay development time will be minimal. Generic solid-phase extraction protocols can also be effective, but will experience a higher (10 to 15% of compounds) failure rate. Chapter 6 contains examples of generic extraction protocols that can be adapted for use with cassettes containing diverse NCEs. 

The specification of the samples (serum, plasma or urine), sampling method, volume and number of samples should be stated in the clinical trial protocol and the information provided to the volunteer. In the case of plasma samples the anticoagulant to be used should be specified in the protocol. 

FIGURE1.4 A comparison of three extraction protocols for the analysis of rat plasma samples for propranolol. (Reprinted from Mallet, C.R. et al., Rapid Commun. Mass Spectrom., 17, 163, 2003. With permission.)... 

Perhaps a better approach than this arbitrary dose-driven escalation is pharmacokinetic or drug concentration-driven dose escalation. This requires flexibility in protocol design, rapid assay of plasma samples, and pharmacokinetic analysis of results between dosing escalations. However, there are many advantages with this approach. Issues related to bioavailabihty or nonlinear pharmacokinetics are immediately apparent and can be readily addressed. Selecting subsequent doses based on pharmacokinetic data from previous doses increases both safety and efficiency fewer dose escalation steps are usually required. Another potential advantage is better correlation with safety and pharmacological effects. 

Sample concentration is another way to increase the sensitivity of a method. Besides liquid-liquid and solid-phase extraction protocols, which result in more concentrated solutions in addition to sample cleanup, several electrokinetic concentration techniques can be applied including sample stacking and field-amplified sample injection/stacking. Over 1000-fold sensitivity enhancement compared to hydrodynamic sample injection without stacking has been reported. When acetonitrile has been used for the removal of proteins from plasma, sample injections of up to 50% of the capillary volume become possible. Stacking techniques are also available for MEKC. 

The major steps in the protocol for the assay described include (I) preparation of serum or plasma samples for analysis by solid phase extraction, and (2) analysis of samples by LC-MS/MS. 

Newly absorbed carotenoid is most easily detected in the chylomicron or TRL plasma fraction, even in the absence of a whole-plasma response (Faulks etal. 1997) partly because this fraction is small and devoid of carotenoid in fasting volunteers, it is the most responsive pool and does not contain carotenoids sequestered and re-exported by the liver. The only disadvantage in this method is the necessity of obtaining fairly large plasma samples (5 ml) and the time taken to density adjust the plasma, ultracentrifugation and the quantitative recovery of the chylomicron fraction. A shorter and less demanding protocol for the isolation of chylomicrons has now been described (Borel et al. 1998). 

The sample materials from which proteins for proteomics studies may be extracted include fresh or snap-frozen cells from varied sources such as biological fluids, (serum, urine, plasma) and solid tissues such as biopsy specimens. Moreover, proteins isolated from ethanol-fixed paraffin-embedded tissues can be utilized for MS analysis.2 Protocols for the identification of proteins from formalin-fixed paraffin-embedded (FFPE) tissues have been recently developed.3 4 FFPE materials are the most common forms of biopsy archives utilized worldwide, and represent an important advancement for the large-scale interrogation of proteins in archival patient-derived materials. Finally, laser capture microdissected tissues have been successfully used for MS analysis.45... 

Isobaric interferences (especially those arising from the plasma itself, e.g., ArO+ on Fe) can be eliminated using cool-plasma conditions, sometimes in combination with a shield torch. This option is not suitable for seawater samples because a cool plasma, in the presence of a heavy matrix, cannot fully ionize elements with high first ionization potentials, notably Zn, Cd, and Hg. Protocols have thus been established for analysis of 10-fold diluted seawater on instalments with sufficiently high resolution to separate most of the affected isotopes from their isobaric interferences [1], To circumvent the issue entirely, others have used online chemical extraction to separate analytes of interest... 

Pharmacokinetic concentration-time curves for a drug and ifs mefabolifes are used to identify primary exposure metrics such as AUC, or which are not time-dependent unlike the sequential measurements of concentration over time. A peak plasma concentration of a drug is often associated with a PD response, especially with an adverse event. There can be large inter-individual variability in the time-to-peak concentration, and closely spaced sampling times are often critical to determining the peak plasma concentration accurately in individual patients because of differences in demographics, disease states, and food effects, if any. All these elements are clearly spelled out in the protocols written to conduct these studies. 

Commercial kits are available for DNA extraction from the majority of sources, including blood, mouthwash, plasma, serum, frozen tissue, and formalin-fixed tissue. Most kits work very weU if carried out in accordance with the manufacturer s instructions. In addition, they reduce the hkeh-hood of variabihty in the quality and quantity of DNA between batches of samples. Published protocols for the extraction of DNA from paraffin-embedded tissue are also available (37). 

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