Freeze-thaw analysis

A typical outcome of the Helfrich analysis for charged bilayer systems is presented in Figure 24b-d in combination with experimental results for the dependence of the equilibrium radius of vesicles composed of DOPG and DOPC as a function of the ionic strength in Figure 24a. The vesicle solution was equilibrated using the freeze-thaw method [20], and the size was measured... 

Compounds frozen in DMSO are generally stable. However, the number of ffeeze/thaw cycles must be kept to a minimum to avoid compound loss. Compound loss is mainly due to precipitation, whereas compound loss due to degradation is negligible (no additional peaks are detected in the HPLC analysis). It has been reported that 10 M stock solutions can undergo more than 10 freeze/thaw cycles with no significant effect on compound stability. Long-term storage of compounds at room temperature in DMSO, over a three-month period, is also possible with only minor loss of compound. Kozikowski etal observed a less than 20% loss after three months in 92% of the cases. 

Catalytic H2O2 Oxidations. The reactions were carried out in 10-mL tubes equipped with serum cap and a stirring bar. The catalyst, hydrogen peroxide, and substrate were dissolved in of CH3CN. Trimethylacetonitrile was added to the reaction as an internal standard. All reactions were done under argon, each was purged by three freeze-thaw cycles and GC analysis was performed on aliquots withdrawn directly from the reaction mixture. Typically, alkene (1 mmol) was added... 

Figure 11.9 Schematic view of the experimental strategy for carrying out poly(Phe) synthesis in POPC liposomes, (i) Freeze-thaw (x 7) solution containing t-RNAP , poly(U), Phe, ATP, GTP, Mg(OAc)2, NH4CI, spermine, spermidine, phos-phoenolpyruvate. (ii) Soution containing pyruvate kinase, 100 000 g supernatant enzymes, 308 and SOS ribosomal subunits, (iii) 1. Free-thaw (x3) 2. Brief extrusion 3. Addition of EDTA (final concentration = 35 mM. (iv) Withdrawl of aliquots at indicated time and cold TCA precipitation. Analysis of the radioactivity remaining on the glass filter by p-scintillation counting. (Modified from Oberholzer et al, 1999.)...

A random urine sample can be used for the analysis of polyols. TALDO and RPI deficiencies, as well as galactosaemia and presumably sorbitol dehydrogenase deficiency, can be diagnosed using urine. The samples are stored frozen. Repeated freeze-thawing does not influence the concentrations of the polyols. 

Stability of samples prior to and during analysis is an important consideration when developing and validating an analytical method. For analysis of CCA, Souri et al. [42] reported that the stability of CCA in rat plasma samples was up to 48 days, or 3 cycles of freeze-thaw, when stored at - 70 5 °C. When stored at ambient temperature (20-25 °C),... 

Frerichs et al. [128] developed and validated a method for the quantitation of omeprazole and hydroxyomeprazole from one 250 [A sample of human plasma using HPLC coupled to tandem mass spectrometry. The method was validated for a daily working range of 0.4-100 ng/ml, with limits of detection between 2 and 15 pg/ml. The interassay variation was less than 15% for all analytes at four control concentrations and the samples were stable for three freeze-thaw cycles under the analysis conditions and 24 h in the postpreparative analysis matrix. The method was used to analyze samples in support of clinical studies probing the activity of the cytochrome P-450 enzyme system. 

Antidepressant stability after three freeze-thaw cycles was evaluated in triplicate at low and high concentrations. Calculated concentrations in the samples subjected to these conditions (stability samples) were compared to those obtained in freshly prepared samples (control samples). Stability and control samples were quantified with a calibration curve prepared on the day of the analysis. All analytes were stable under these conditions, except sertraline, for which a slight signal decrease was observed at 250 ng/mL in oral fluid (MRE = -33.4% %CV = 6.0%). 

Fig. 3. Activity of HRP following LEF induced uptake into COS 5-7 and HaCaT cells. Cells (2 x 106/mL) were exposed to LEF (20 V/cm, 500 Hz, and pulse width 180 (is) for 1 min in the presence of 1 mg/mL HRP in DMEM-H medium. Three hours after exposure, the cells were washed three times in PBS and disrupted by five freeze-thaw cycles in 50 (jlL PBS containing anti-protease cocktail (1 100). Dot-blot analysis of HRP activity 1 - cells without HRP 2 - non-exposed cells with HRP 3 and 4 -duplicates of cells exposed to LEF in the presence of HRP.

The assay has been validated and the results of validation demonstrate that the standard curve is linear over the concentration range of 100-2000 ng/mL. The assay is reproducible and accurate, with recovery of the analyte and internal standard in the range of 80-90 %. The analysis requires 0.5 mL of plasma and has a limit of quantification of 70 ng/mL. The stability of plasma samples stored at -20 °C has been demonstrated for up to 12 weeks. Autoinjector stability has been demonstrated for over 13 h and freeze-thaw stability has been demonstrated for 3 freeze-thaw cycles. The procedure has a sample throughput of at least 30 specimens per day. The assay meets the guidelines for bioanalytical methods validation for human studies (Shah et al. 1991). 

Figure 6 Schematic of a complete multiplexed and integrated instrumental design for PCR analysis with eight channels. Different functional capillaries were connected by a T-assembly and a cross-assembly. Stars represent the freeze/thaw valves V1 and V2. A syringe pump and a six-way selection valve were used to load and distribute liquids for various purposes. A CCD camera was used to collect fluorescence from the eight capillaries simultaneously. The temperature control unit at the cross-assembly was used to denature DNA prior to injection. (Reprinted from Ref. 34 with permission.)...

The methods used to correlate suspensibility may be used with other response variables — such as biological activity, stability, or yield value — that are important in the development of pesticides. Also, formulations of greater complexity, which have more components, can be studied with the same correlational techniques. There are, however, many important response variables that are qualitative or semiquantitative and are not usually estimated with the precision necessary for reliable regression analysis. These include, freeze-thaw stability, bloom, compatibility, and... 

Bacteria that remained viable after 48 freeze-thaw cycles were used to initiate new cultures and these were subjected to additional freeze-thaw cycles as described. Individual isolates, which had been previously identified, as well as the control cultures, were also subjected to freeze-thaw cycles. Occasionally, single isolates in 10% TSB would supercool rather than freeze at temperatures close to 0°C, and to ensure that all samples froze at the same temperature, a few sterilized Agl crystals were added to the vials at the start of the experiments. For some experiments, cells were harvested by centrifugation (6,000 xg for 10 min) and the cell pellet washed with 10% TSB and kept at 0°C until analysis. Spent media was obtained by centrifuging, as above, and filtering (0.45 pm). 

Gika et al. [98,99] investigated short-term sample stability for human urine by LC-MS. Urine samples were collected from normal female and male volunteers into containers containing one protease tablet and frozen at -80°C or -20°C for one month. Upon defrosting, samples were centrifuged at 13,000rpm for lOmin and then diluted 1 4 with 0.1% formic acid for LC-MS analysis. All samples were kept at 4°C in an autosampler. LC-MS analyses showed that there were no differences observed between samples stored at -80°C or -20°C even after 1 month. Therefore, storing urine samples at -20°C for at least one month is acceptable. In addition, based on principal component analysis (PCA) the data obtained from samples that had been subjected from one to nine freeze-thaw cycles showed that overall stability of the samples was also acceptable. 

Changing tips between pipetting of each QC set also improves overall QC accuracy. Also check that a new QC lot has been properly qualified. Other questions to consider include the following Are the calibrators and the QCs prepared in the same lot of pooled matrix If not, do the two matrix pools exhibit comparable spike recovery of the analyte Are the calibrators and QCs prepared from different lots of source material If yes, check respective certificates of analysis for purity. Is there a sensitivity to freeze/ thaw Check for validated stability of QC samples against storage conditions. Have the standard calibrators and QC samples experienced the same number of freeze/thaw cycles Answers to these questions often lead to the determination of the causes of failures. 

The freeze thaw stability evaluation should take into account the anticipated freeze thaw cycles during routine sample analysis. A standard approach is four freeze thaw cycles with no less than 12 h frozen storage between unassisted thaws. The rate of freezing and thawing should take into account the known properties of the therapeutic as well as the manner in which samples will be handled as they are being thawed before assaying. For example, labile molecules may require a slow thawing process on ice. 

We also tend to collect a volume that is large enough to provide all the aliquots to be stored at different temperatures and conditions, and then store those aliquots at the conditions required, placing the aliquots into the — 80°C freezer when each evaluation at the selected time period and temperature has been completed. Hence, we build up, over time, a stock of aliquots stored at —80°C that have been potentially stored at —80°C, — 20°C, 4°C, room temperature, freeze-thaw cycles, and so on, and then thaw and analyze all these samples in one batch to reduce batch-to-batch variability. However, this also requires the analysis of the original sample before freezing as immediately as possible after collection. This is the zero-time sample, the concentration of which is the baseline value that future results of the various stored samples are measured against to prove stability. 

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