Sample stabilization strategies: a case study review of unique sample collection and handling procedures.

The ability to maintain analyte stability is crucial in order to obtain accurate and meaningful data. Stability should be evaluated in a manner that the entire lifecycle of the sample is taken into account, that is, from the moment the sample is collected from the patient until the last analysis is complete. Evaluation of the sample collection and handling procedure is thus necessary in order to prevent analyte instability potentially encountered downstream in the analytical process. This manuscript details cases of analyte instability attributed to factors such as enzymatic degradation, matrix composition, analytical technique and oxidation to name a few. As well each case describes a unique collection and handling procedure that was necessary to achieve stability prior to analysis.

Very complex internal standard response variation in LC-MS/MS bioanalysis: root cause analysis and impact assessment.

Internal standards (ISs) are essential for the development and use of reliable quantitative bioanalytical LC-MS/MS methods, because they correct for fluctuations in the analytical response that are caused by variations in experimental conditions. Sample-to-sample differences in the IS response are thus to be expected, but a large variability often is an indication of nonoptimal sample handling or analysis settings. This paper discusses a number of cases of very complex variation of IS responses that could be attributed to analytical problems such as injection errors and sample inhomogeneity, and matrix-related issues such as degradation and increased ionization efficiency. A decision tree is proposed to help find the underlying root cause for extreme IS variability.

Assay development for determination of tenofovir in human plasma by solid phase analytical derivatization and LC-MS/MS.

BACKGROUND: A novel method was developed and validated to measure tenofovir in human plasma. RESULTS/METHODOLOGY: This method employed solid phase analytical derivatization and analysis by LC-MS/MS. Stable-labeled internal standard was added to plasma samples followed by solid phase extraction. Retained analytes were derivatized on the solid phase extraction cartridges with a diazomethane solution to yield methyl-ester derivatives. Samples were analyzed using LC-MS/MS incorporating the use of a strong cation exchange column. The method was validated over a range of 5.00-750 ng/ml. The approach developed in this report for tenofovir could be applied to other analytes that share similar structural similarities. CONCLUSION: The tenofovir LC-MS/MS method was used to support a clinical study of over 400 samples with a 100% success rate.

Kinetic studies of drug-protein interactions by using peak profiling and high-performance affinity chromatography: examination of multi-site interactions of drugs with human serum albumin columns.

Carbamazepine and imipramine are drugs that have significant binding to human serum albumin (HSA), the most abundant serum protein in blood and a common transport protein for many drugs in the body. Information on the kinetics of these drug interactions with HSA would be valuable in understanding the pharmacokinetic behavior of these drugs and could provide data that might lead to the creation of improved assays for these analytes in biological samples. In this report, an approach based on peak profiling was used with high-performance affinity chromatography to measure the dissociation rate constants for carbamazepine and imipramine with HSA. This approach compared the elution profiles for each drug and a non-retained species on an HSA column and control column over a board range of flow rates. Various approaches for the corrections of non-specific binding between these drugs and the support were considered and compared in this process. Dissociation rate constants of 1.7 (±0.2) s(-1) and 0.67 (±0.04) s(-1) at pH 7.4 and 37°C were estimated by this approach for HSA in its interactions with carbamazepine and imipramine, respectively. These results gave good agreement with rate constants that have determined by other methods or for similar solute interactions with HSA. The approach described in this report for kinetic studies is not limited to these particular drugs or HSA but can also be extended to other drugs and proteins.

Measurement of drug-protein dissociation rates by high-performance affinity chromatography and peak profiling.

The rate at which a drug or other small solute interacts with a protein is important in understanding the biological and pharmacokinetic behavior of these agents. One approach that has been developed for examining these rates involves the use of high-performance affinity chromatography (HPAC) and estimates of band-broadening through peak profiling. Previous work with this method has been based on a comparison of the statistical moments for a retained analyte versus nonretained species at a single, high flow rate to obtain information on stationary phase mass transfer. In this study an alternative approach was created that allows a broad range of flow rates to be used for examining solute-protein dissociation rates. Chromatographic theory was employed to derive equations that could be used with this approach on a single column, as well as with multiple columns to evaluate and correct for the impact of stagnant mobile phase mass transfer. The interaction of L-tryptophan with human serum albumin was used as a model system to test this method. A dissociation rate constant of 2.7 (+/-0.2) s(-1) was obtained by this approach at pH 7.4 and 37 degrees C, which was in good agreement with previous values determined by other methods. The techniques described in this report can be applied to other biomolecular systems and should be valuable for the determination of drug-protein dissociation rates.

Analysis of free drug fractions using near-infrared fluorescent labels and an ultrafast immunoextraction/displacement assay.

A chromatographic method was developed for measuring free drug fractions based on the use of an ultrafast immunoextraction/displacement assay (UFIDA) with near-infrared (NIR) fluorescent labels. This approach was evaluated by using it to determine the free fraction of phenytoin in serum or samples containing the binding protein human serum albumin (HSA). Items considered in the design of this method included the dissociation rate of HSA-bound phenytoin, the rate of capture of free phenytoin by immunoextraction microcolumns, the behavior of NIR fluorescent labels in a displacement format, and the overall response and stability of the resulting assay. In the final UFIDA method, the free fraction of phenytoin was extracted in approximately 100 ms by a microcolumn containing a small layer of anti-phenytoin antibodies. This gave a displacement peak for a NIR-fluorescent-labeled analogue of phenytoin that appeared within 2-3 min of sample injection, creating a signal proportional to the amount of free phenytoin in the sample. The UFIDA method provided results within 1-5% of those determined by ultrafiltration for reference samples. The lower limit of detection was 570 pM, and the linear range extended up to 10 microM. This approach is not limited to phenytoin but can be adapted for other analytes through the use of appropriate antibodies and labeled analogues.

Studies by biointeraction chromatography of binding by phenytoin metabolites to human serum albumin.

Biointeraction studies based on high performance affinity chromatography were used to investigate the binding of human serum albumin (HSA) to two major phenytoin metabolites: 5-(3-hydroxyphenyl)-5-phenylhydantoin (m-HPPH) and 5-(4-hydroxyphenyl)-5-phenylhydantoin (p-HPPH). This was initially examined by conducting self-competition zonal elution experiments in which m-HPPH or p-HPPH were placed in both the mobile phase and injected sample. It was found that each metabolite had a single major binding site on HSA. Competitive zonal elution experiments using l-tryptophan, warfarin, digitoxin, and cis-clomiphene as site-selective probes indicated that m-HPPH and p-HPPH were interacting with the indole-benzodiazepine site of HSA. The estimated association equilibrium constants for m-HPPH and p-HPPH at this site were 3.2 (+/-1.2)x10(3) and 5.7 (+/-0.7)x10(3)M(-1), respectively, at pH 7.4 and 37 degrees C. Use of these metabolites as competing agents for injections of phenytoin demonstrated that m-HPPH and p-HPPH had direct competition with this drug at the indole-benzodiazepine site. However, the use of phenytoin as a competing agent indicated that this drug had additional negative allosteric interactions on the binding of these metabolites to HSA. These results agreed with previous studies on the binding of phenytoin to HSA and its effects on the interactions of HSA with site-selective probes for the indole-benzodiazepine site.

Studies of phenytoin binding to human serum albumin by high-performance affinity chromatography.

High-performance affinity chromatography was used to study the binding of phenytoin to an immobilized human serum albumin (HSA) column. This was accomplished through frontal analysis and competitive binding zonal elution experiments, the latter of which used four probe compounds for the major and minor binding sites of HSA injected into the presence of mobile phases containing known concentrations of phenytoin. It was found that phenytoin can interact with HSA at the warfarin-azapropazone, indole-benzodiazepine, tamoxifen, and digitoxin sites of this protein. The association constants for phenytoin at the indole-benzodiazepine and digitoxin sites were determined to be 1.04 (+/-0.05) x 10(4)M(-1) and 6.5 (+/-0.6) x 10(3)M(-1), respectively, at pH 7.4 and 37 degrees C. Both allosteric interactions and direct binding for phenytoin appear to take place at the warfarin-azapropazone and tamoxifen sites. This rather complex binding system indicates the importance of identifying the binding regions on HSA for specific drugs as a means for understanding the transport of such substances in blood and in characterizing their potential for drug-drug interactions.

Characterization of drug interactions with soluble beta-cyclodextrin by high-performance affinity chromatography.

This study examined the use of an immobilized human serum albumin (HSA) column to study solution-phase reactions between drugs and beta-cyclodextrin (beta-CD). Chromatographic equations were developed to characterize the binding of chemicals to a soluble ligand (beta-CD) in the presence of an independent immobilized ligand (HSA). Situations considered included the presence of both a homogeneous and heterogeneous immobilized ligand, as well as complex interactions between the chemical of interest and soluble ligand. Three drugs (warfarin, tamoxifen, and phenytoin) were examined by this approach. This method involved injecting a small amount of each drug onto an HSA column in the presence of various concentrations of beta-CD in the mobile phase. By measuring the change in the drug's retention factor as the concentration of beta-CD was varied, it was possible to determine the stability constant between the injected drug and beta-CD. With this approach, warfarin and beta-CD were found to have 1:1 interactions with a stability constant of 5.2 x 10(2) M(-1) at 37 degrees C and pH 7.4, a result in close agreement with previous literature values. Tamoxifen and phenytoin were also found to have 1:1 interactions with beta-CD and had stability constants of 0.9-1.2 x 10(4) and 6-9 x 10(2) M(-1) respectively. With these latter solutes, the effects of secondary binding to the chromatographic support had to be considered. The theory and methods described in this report are not limited to these drugs and beta-CD but can be applied to other analytes and soluble ligands.