Freeze thaw sample stability

Freeze and Thaw Stability. Analyte stability should be determined after three freeze-thaw cycles. At least three aliquots at each of the low and high concentrations should be stored at the intended storage temperature for 24 h and thawed unassisted at room temperature. When thawed completely, the samples should be refrozen for 12 to 24 h under the same conditions. The freeze-thaw cycle should be repeated twice more, then analyzed on the third cycle. If an analyte is unstable at the intended storage temperature, the stability sample should be frozen at —70°C during the three freeze-thaw cycles. 

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),... 

The stability of etodolac in equine serum and urine was studied under various temperatures (freezing, refrigerated, and room temperatures) for up to 45 days [32]. Etodolac was stable in urine and serum under frozen condition for 45 days, at refrigerated temperature for 30 days (7 days for urine). At room temperature, etodolac was stable in serum for up to 7 days, whereas it was stable in urine for 2 days. Etodolac serum and urine samples also showed stability through 5 freeze-thaw cycles. 

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%). 

All analytical methods must be validated in accordance with international guidelines [59-61] prior to application. As discussed in the first part of this book, minimum criteria that need to be met to in order to satisfy these guidelines are selectivity, matrix effects, extraction efficiency, process efficiency, processed sample stability, linearity, accuracy, precision, and freeze-thaw stability. 

Sample stability 3x Freeze-thaw cycles for samples between -70 °C and room temperature 4-24 h at room temperature long-term storage sample stock and postpreparation stability. 5-10 ng/Lx6 at <6 °C for 28 days NA 3x Freeze-thaw cycles for samples between -20 °C and room temperature Ambient and 4 °C for 7 days... 

Human specimens. In our laboratory, we first developed a good laboratory practice (GLP)-validated procedure for quantification of intact rafAON in control human plasma. The rafAON assay validation endpoints were standard curve, between-run precision and accuracy, within-run precision and accuracy, effects of dilution and freeze thaw, stability of rafAON at -80° C, and 4°C in plasma for various times, specificity, integrity of rafAON during plasma sample collection and processing, and lipid interference. The reader is referred to a previous citation for further details (17,27). 

Tests on sample stability, carry over, specificity, matrix interference, sample stability (freeze/thaw stability and so forth)... 

Sample stability was also assessed. No stability issue was found when spiked brain homogenates were stored at room temperature (4 h), or in the processed samples over 48 h. The stability of hyperforin was also tested over three freeze-thaw cycles, which again did not reveal any significant degradation of the drug. 

Sample stability was also assessed. Both compounds (simvastatin and simvastatin acid) were stable for at least 2 months at -70 °C and also for at least three freeze-thaw cycles. Processed samples also proofed stability over 24 hours at 4°C. Some degradation of simvastatin was found within 24 hours at room temperature (between 23 and 39 %). For simvastatin acid on the other hand, the found concentration increased within 24 h at room temperature (+ 23 to + 29 %), indicating that a fraction of simvastatin was hydrolyzed within 24 h at room temperature. 

Autoinjector stability was carried out for over 13 h by repeated injection of the same extracted plasma sample at room temperature (nominally 25 °C) The results showed, that the extracted specimens remained stable over the course of the study. QC samples containing 120 and 2000 ng/mL DEC in plasma were subjected to 3 freeze/thaw cycles. Samples were frozen at -20 °C for 24 h and thawed unassisted at room temperature. 

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). 

To achieve optimum compound stability, aUquoted compound solutions for HTS are stored in a solid solvent matrix (frozen water-free DMSO). Thereby, repetitive freeze-thaw cycles of the compound solutions in the aliquoting and sample distribution processes can be avoided. The sample plates for a screening campaign are transferred sealed and frozen to the HTS lab. 

The Liquid Master Store at Roche uses a 96-tube version of the microtube plate described in Section 1.4 a description of the 96-tube plate is found on the web site www.remp.com. The individually accessible tubes in 96-tube plates in the Liquid Master Store hold a few definite sample volumes, e.g. 26 J,L and 140 J,L per tube. Storage and retrieval of HTS master solutions with fixed sample volumes facilitate the sample logistics considerably. Upon retrieval from the Liquid Master Store, the master solutions in the 96-tube plates are thawed and transferred to 384-well plates before they enter the downstream aliquoting process. This sample-handling concept avoids repetitive freeze-thaw cycles of HTS master solutions and thereby improves the stability and integrity of the compound samples used in HTS. 

This is a useful guide to the physical stability of suspensions. If freeze—thaw cycling or elevated temperature exposures are chosen for physical stability testing, companion samples of a closely related marketed suspension should be included in the testing protocol for comparison purpose because pharmaceutical suspensions are not normally designed to withstand temperature extremes during storage (15-30°C optimum). 

As samples are often frozen and thawed, e.g. for re-anal-ysis, the stability of analyte during several freeze/thaw cycles should also be evaluated. The Conference Reports require a minimum of three cycles at two concentrations in triplicate, which has also been accepted by other authors [2, 4, 6, 9,10]. 

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