Mass balance assay

Figure 4.42. Trend analysis over 46 batches of a bulk chemical produced according to the same manufacturing procedure Small and scaled-up batch size [kg], HPLC and Titration assays [%], resp. individual HPLC impurity levels [%], versus batch number. The lack of full correlation between assays indicates that the titration is insensitive to some impurities detected by HPLC. The mass balance, where available, suggests that all relevant impurities are quantified. Impurities B and C, for instance, are highly correlated (r = 0.884, p = 0.0002).

Hydrocarbon-degrading microorganisms are ubiquitous in most ecosystems [32,117] however, it is often very difficult to prove that transformation, degradation, and mineralization actually occur in the environment because it is difficult to distinguish contributions from biotic and abiotic processes under uncontrolled conditions in the natural environment [338]. In contrast, laboratory assays can provide definitive evidence for microbial degradation, and sterilized samples can be used to determine abiotic losses. Thus, contributions from microbial degradation can be differentiated from abiotic loss by a mass balance... 

To estimate the recovery of the protein from the capillary, a sample was labeled with 125I to track mass balance. A nonradiolabeled sample was used to show that both samples produced the same CE profile. A baseline value or control was obtained by performing the assay through the injection step, followed by a high-pressure rinse to expel the entire capillary contents into a collection vial. A... 

The sample must be soluble If it s not in solution, it cannot be analyzed by HPLC. Although this may seem obvious, solubility issues complicate real assays of low-solubility drugs and controlled-release formulations. Many situations encountered in pharmaceutical analysis, such as low recovery, lack of mass balance, and out-of-specification results, might stem from solubility problems in a sample preparation step, rather than the HPLC analysis itself. 

In this study, field, petrographic, bulk geochemical and mineral chemical data are presented with the aim of elucidating the mineralogical residence and spatial distribution of Ag within the HRMZ. A mass balance was calculated for Ag from electron microprobe analyses of sulfide minerals relative to bulk assays for the Main Zone deposit previously published by Wardrop (2006) for Sabina Silver Corporation. 

Table 1. Mass balance of silver in minerals from the Hackett River Main Zone relative to the Ag bulk assays from Wardrop (2006).

Any evaluation should consider not only the assay but also the degradation products and other appropriate attributes. Where appropriate, attention should be paid to reviewing the adequacy of the mass balance and different stability and degradation performance. 

Viral detection assays based on infectivity suffer from significant variability, which necessitates the use of statistical evaluation. Polymerase chain reaction-based assays are currently being developed and validated for viral clearance. With PCR assays, there is a potential to distinguish between inactivation and physical removal, perform mass balance studies, evaluate more than one vims at a time for a given process step, reduce the time for completing clearance studies, and accurately quantitate the amount of vims bound to such surfaces as chromatography resins. Table 5 compares the assay precision between an infectivity assay and a quantitative PCR assay. 

Condition Gemcitabine assay (% Initial) Related substances (%) Mass balance (%) Relative Mass Balance deficita(%)... 

The assessment of degradation in pharmaceutical products involves two aspects of analytical measurement. First, a selective analytical method must be available for accurate assay of the parent drug compound, in order to correctly measure any loss. Second, methodology should be in place for quantification of the degradation products formed. Ideally, when degradation occurs, the measured amount of parent drug lost should correlate well with the measured increase in degradation products. This correlation is referred to as mass balance )- More recently, the International Conference on Harmonization (ICH) has provided a definition of mass balance material balance as follows ... 

Kirschbaum JJ. Synergistic use of multiple assays and achievement of mass balance to validate analytical methods. Trends Anal Chem 1988 7 16-20. 

The main variable of design for a CSTR is the hydraulic retention time (HRT), which represents the ratio between volume and flow rate, and it is a measure of the average length of time that a soluble compound remains in the reactor. Capital costs are related to HRT, as this variable directly influences reactor volume [83]. HRT can be calculated by means of a mass balance of the system in that case, kinetic parameters are required. Some authors obtained kinetic models from batch assays operating at the same reaction conditions, and applied them to obtain the HRT in continuous operation [10, 83, 84]. When no kinetic parameters are available, HRT can be estimated from the time required to complete the reaction in a discontinuous process. One must take into account that the reaction rate in a continuous operation is slower than in batch systems, due to the low substrate concentration in the reactor. Therefore, HRT is usually longer than the total time needed in batch operation [76]. 

If 2 or more filters do not meet acceptance criteria compound assay should be repeated (exception compounds with unusual features causing for example a discrepancy in mass balance due to high plastic binding). 

This type of hADME studies using radioactive-labeled drag should be run unless phase I studies show that > 90 % of the dose is excreted unchanged in urine. In this case, a hADME study may not be required. For those drags, a urine assay should be the prerequisite for phase I trials and then mass balance may be established in trials where quantitative urine collections are performed. If phase I data indicate that cold mass balance , i.e. >90% recovery in urine cannot be obtained, a hADME study has to be scheduled for the development program. 

Whole-animal studies assess the percent of the applied dose absorbed into the body using classic techniques of bioavailability, where absorbed chemical is measured in the blood, urine, feces, and tissues with mass balance techniques. Recently, methods have been developed to assess absorption by measuring the amount of chemical in the stratum comeum because it is the driving force for diffusion. Cellophane tape strips are collected 30 minutes after chemical exposure and the amount of drug assayed in these tape strips correlates to the amount systemically absorbed. If the focus of the research is to determine the amount of chemical that has penetrated into skin, core biopsies may be collected and serially sectioned, and a profile of the chemical as a function of skin depth may be obtained. 

In this case a Toepler pump can be used to remove the volatile products derived from a known mass of the gallane, and an efficient trap cooled to 77 K to separate the condensable B2H6 from the noncondensable H2 the two fractions are then assayed tensimetrically. The mass balance is completed simply by weighing the residue of elemental gallium. Similar measures can be adopted to determine the stoicheiometry of a reaction engaging a gallane with another compound, e.g., NH3, NMe3, or HC1. 

The next study was conducted in a fashion identical to the initial study in HDPE bottles, except the tablet was placed inside the capsule (overencapsulated tablet) with the tablets as a control experiment. Sure enough, the tablets that were not over-capsulated showed a mass balance issue, while the same batch of tablets inside a capsule showed no mass balance issue. This is very interesting, since the capsule is preventing the loss of assay value. The next obvious experiment was to study the packaging material. The experiment was set up such that tablets were stored in an amber glass bottle and HDPE induction seal bottles for 6 weeks at 40°C/75% RH. In addition, over-encapsulated tablets were stored in HDPE induction seal bottles for 6 weeks at 40°C/75% RH. Results are shown in Figure 15-17. It can be concluded from the data presented in Figure 15-17 that the active was possibly adsorbed to the... 

However, a SIM during formulation development involves more than the above four steps, since the formulation development is a dynamic process where the formulation is optimized as the clinical program moves further along the development process. To continue the list from above, the following three steps must be added to the Ust (5) compatibility studies with excipients, (6) stability trending (variables would be temperature, humidity, and packaging material), and (7) mass balance for assay. An analytical chemist must revisit the separation of all components in the related substances method after steps 3,5, and 6 to ensure that the test method is truly a SIM. 

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