**3. NCS stability study experience**

The overall objective of the planned NCS storage studies was to determine the stability of target analytes in NCS environmental samples stored in collection containers at specific storage conditions, including reconstitution after thawing and re-freezing of samples, as applicable. For instance, the objective of the tap water study was to evaluate the effect of prolonged storage at −20°C on the stability of the pesticide and pharmaceutical target analytes spiked in analyte-free water. The results of this study were to be used to extrapolate the observed changes in the target analyte concentrations to the stability of those compounds in the NCS study samples.

#### **3.1 Planning**

The stability study plans included stability samples similar to environmental field samples, sample processing, analytes or classes of analytes of interest, and analysis methods. The in-depth literature review addressed the prevalence of

*Analytical Chemistry - Advancement, Perspectives and Applications*

**208**

**Matrix**

**Analytes** N-methylcarbamates in beef,

HPLC

duck, chicken liver

**Analysis method(s)**

**Temperature**

−4 °C

0.5, 1, 1.5, 2, 3,

4, 5, 6 mo

*AAS – Atomic absorption spectrometry, AES – Atomic emission spectrometry, APCI – Atmospheric pressure chemical ionization, DAD – Diode array detector, ECD – Electron capture detection, EDC -* 

*Electrostatic dust collector, ELISA - Enzyme-linked immunosorbent assay, FID – Flame ionization detection, GC – Gas chromatography, HPLC – High pressure liquid chromatography, ICP – Inductively* 

*coupled plasma, LAL – Limulus amoebocyte lysate, LC – Liquid chromatography, MS – Mass spectrometry, NPD - Nitrogen phosphorous detection, SIM - Selective ion monitoring, SRM – Selected reaction* 

*monitorin.*

**Table 1.**

*Summary of environmental sample stability studies identified in the literature by matrix and analytes.*

**Time**

**Other**

**Storage conditions**

**Results** Store at cryogenic conditions

[81]

for preparation and storage due

to enzymatic activity. Some

analytes stable for 6 mo, some

not at all

**References**

potential analytes and classes of analytes of interest in an indoor environment, concentrations measured, sampling and analytical methods, reaction or degradation products in these matrices, and storage conditions and stabilities. In addition, experts, agencies and groups with relevant experience, e.g., National Institute of Standards and Technology (NIST), the U.S. Environmental Protection Agency (EPA), the Children's Center Study, National Human Exposure Assessment Survey (NHEXAS), Children's Total Exposure to Persistent Pesticides and Other Persistent Organic Pollutants (CTEPP), and New York State Department of Health/Love Canal project researchers, were contacted for information. This information was used to develop the plans for the preparation and analysis of the stability samples and archiving of both the field and stability samples.

Storage effects due to freeze–thaw cycles, sample pretreatment, and reconstitution were also considered. For example, freeze–thaw cycles can affect the volatile and semi-volatile compound concentrations on a filter or in bulk dust. The pretreatment of samples before storage is performed when metabolism or reaction after collection is known to be of concern. For example, water samples for metals analysis are usually acidified to pH < 2 [86], and dust samples are sometimes irradiated to stop microbial growth and metabolism.

#### **3.2 Acquisition of samples**

Stability study samples with known concentrations of analytes were planned to be acquired in similar matrices to the NCS samples, i.e., particulate filters, wipes, and vacuum filters. These samples were to include (1) newly-prepared samples that have been spiked with known concentrations of analytes, and (2) pre-tested 'realworld' samples.

Surface dust wipe and air diffusive samples could be made by spiking study sampling media and tap water samples by spiking analyte-free water with known amounts of the target analytes. These kind of samples can be purchased from reference material producers, proficiency testing sample providers, and/or accredited laboratories. To allow for a possible difference in reaction/degradation rate of analytes with their concentration in matrix, each matrix was to be spiked at environmentally relevant concentration levels, based on the literature review, and at concentrations that can be most reliably quantified by the analytical method employed, preferably at concentrations near the midpoint of the instrumental calibration range. Another benefit of this approach was that the concentration of the stability samples could be set to match the concentration used in the laboratory QC samples for increased comparability [7].

Some samples cannot be spiked onto matrices in a manner representative of the corresponding environmental samples, or it would be very difficult to do so. There may be a difference in behavior of analytes in environmental samples and in similar spiked matrices due to pH, microbial enzymatic activity of the sample matrix, physicochemical properties of the analytes themselves and their reactions with the matrix, and other analytes present in the environment. For example, for vacuum filter and deposition plate stability samples, dust from non-participant homes could be obtained, homogenized, and aliquoted before storage.

Each spiked and real-world sample should represent the environmental sample matrix as close as possible to account for inter-analyte and analyte-matrix interactions. If it is not possible to include all analytes of interest in one matrix sample, they can be classified into groups by their chemical characteristics, and include representative analytes of each group deemed most unstable. Because sample homogeneity is very important, for example, surface wipe and dust samples should be aliquoted before spiking and the whole sample used for analysis.

**211**

*Considerations for Stability of Environmental Samples in Storage for Long-Term Studies*

All samples were planned to be collected on exactly the same collection media using the same samplers and stored in the same containers as used in the field study and one manufacturer's lot of each media and containers used, whenever possible. Blank media was to be stored together with the samples for each sample type and analysis time point and unused sample media provided to the analysis laboratories, as required. There should be additional samples stored as back-up for sample loss due to, for instance, filter or container breakage, and for repeat analyses. Additional samples should also be stored to account for the possibility that laboratories or analysis methods may change during the study and simultaneous measurements are necessitated. For each sample type, to the extent possible, samples should be analyzed by one laboratory in order to minimize inter-laboratory bias. For NCS, standard laboratory methods were specified for each analysis, identical to those

Samples are usually analyzed before storage (time point 0) to determine analysis

The frequency of time points is chosen to detect the instability of samples and analytes, such as VOC, as early as possible. It is assumed that in most cases, particularly for the organic analytes, the rate of degradation would be highest at the beginning, that is, first order kinetics; this corresponds to a linear change in the log transformed measurement over time [65, 82, 88]. In the NCS stability plans, at each test time t, it is tested statistically with a t-test if a specified percentile of the concentration measurements (the target percentile) will be greater than the specified limit of 80 percent at some future time, assuming stability decreases exponentially (or the log transformed stability decreases linearly) with time. For calculating sample size, it was assumed that the log transformed ratios have a normal distribu-

As the study proceeds there will be accumulating data from prior test times that can also be used to get a more precise estimate of the slope or fit a nonlinear trend. Calculating samples size when using all prior data is more complicated than calculating sample size for a t-test. The calculations depend on the analysis that might be performed. The number of samples depends on: the alpha and target power for a slope of zero; the standard deviation of the measurement error; the standard deviation of the slope factor; the target percentile (80%); and, the timing of the time points. The test-wise alpha and power for no degradation were set to achieve

The standard deviation of the measurement error can be estimated from the variation among replicate samples or aliquots, typically measured as a relative standard deviation or, equivalently, a coefficient of variation (CV). The sample sizes were calculated using the error term calculated from the estimated coefficient of variation of the measurement methods. This model was assumed to be adequate for calculating sample sizes and for a variety of sample collection designs, analyses, and distributional or statistical model assumptions. The planned frequency of time points for the NCS was 0, 1, 2, 3, 5, 8, 13, 21, 32, 48, 72, 108, 160, and 240months, altogether 14 time points (or test times). The numbers of planned stability samples varied by sample type and were mostly around 300. The variation was due to the

tion and the mean log transformed recovery decreases linearly over time.

method recovery, concentrations at the beginning of the study (time 0), and to verify the homogeneity of the aliquots. This will take into account any analyte losses during pre-analysis, such as extraction and processing. The frequency of future time points and the number of samples required for statistical significance at each

*DOI: http://dx.doi.org/10.5772/intechopen.97445*

planned for the field sample analysis.

**3.3 Number of samples and analysis time points**

point should be planned carefully before start of the study.

an overall mean alpha of 5% and an overall power of 95%.

variation of the CV of different analysis methods.

#### *Considerations for Stability of Environmental Samples in Storage for Long-Term Studies DOI: http://dx.doi.org/10.5772/intechopen.97445*

All samples were planned to be collected on exactly the same collection media using the same samplers and stored in the same containers as used in the field study and one manufacturer's lot of each media and containers used, whenever possible. Blank media was to be stored together with the samples for each sample type and analysis time point and unused sample media provided to the analysis laboratories, as required. There should be additional samples stored as back-up for sample loss due to, for instance, filter or container breakage, and for repeat analyses. Additional samples should also be stored to account for the possibility that laboratories or analysis methods may change during the study and simultaneous measurements are necessitated. For each sample type, to the extent possible, samples should be analyzed by one laboratory in order to minimize inter-laboratory bias. For NCS, standard laboratory methods were specified for each analysis, identical to those planned for the field sample analysis.

#### **3.3 Number of samples and analysis time points**

Samples are usually analyzed before storage (time point 0) to determine analysis method recovery, concentrations at the beginning of the study (time 0), and to verify the homogeneity of the aliquots. This will take into account any analyte losses during pre-analysis, such as extraction and processing. The frequency of future time points and the number of samples required for statistical significance at each point should be planned carefully before start of the study.

The frequency of time points is chosen to detect the instability of samples and analytes, such as VOC, as early as possible. It is assumed that in most cases, particularly for the organic analytes, the rate of degradation would be highest at the beginning, that is, first order kinetics; this corresponds to a linear change in the log transformed measurement over time [65, 82, 88]. In the NCS stability plans, at each test time t, it is tested statistically with a t-test if a specified percentile of the concentration measurements (the target percentile) will be greater than the specified limit of 80 percent at some future time, assuming stability decreases exponentially (or the log transformed stability decreases linearly) with time. For calculating sample size, it was assumed that the log transformed ratios have a normal distribution and the mean log transformed recovery decreases linearly over time.

As the study proceeds there will be accumulating data from prior test times that can also be used to get a more precise estimate of the slope or fit a nonlinear trend. Calculating samples size when using all prior data is more complicated than calculating sample size for a t-test. The calculations depend on the analysis that might be performed. The number of samples depends on: the alpha and target power for a slope of zero; the standard deviation of the measurement error; the standard deviation of the slope factor; the target percentile (80%); and, the timing of the time points. The test-wise alpha and power for no degradation were set to achieve an overall mean alpha of 5% and an overall power of 95%.

The standard deviation of the measurement error can be estimated from the variation among replicate samples or aliquots, typically measured as a relative standard deviation or, equivalently, a coefficient of variation (CV). The sample sizes were calculated using the error term calculated from the estimated coefficient of variation of the measurement methods. This model was assumed to be adequate for calculating sample sizes and for a variety of sample collection designs, analyses, and distributional or statistical model assumptions. The planned frequency of time points for the NCS was 0, 1, 2, 3, 5, 8, 13, 21, 32, 48, 72, 108, 160, and 240months, altogether 14 time points (or test times). The numbers of planned stability samples varied by sample type and were mostly around 300. The variation was due to the variation of the CV of different analysis methods.

*Analytical Chemistry - Advancement, Perspectives and Applications*

archiving of both the field and stability samples.

stop microbial growth and metabolism.

QC samples for increased comparability [7].

be obtained, homogenized, and aliquoted before storage.

be aliquoted before spiking and the whole sample used for analysis.

**3.2 Acquisition of samples**

world' samples.

potential analytes and classes of analytes of interest in an indoor environment, concentrations measured, sampling and analytical methods, reaction or degradation products in these matrices, and storage conditions and stabilities. In addition, experts, agencies and groups with relevant experience, e.g., National Institute of Standards and Technology (NIST), the U.S. Environmental Protection Agency (EPA), the Children's Center Study, National Human Exposure Assessment Survey (NHEXAS), Children's Total Exposure to Persistent Pesticides and Other Persistent Organic Pollutants (CTEPP), and New York State Department of Health/Love Canal project researchers, were contacted for information. This information was used to develop the plans for the preparation and analysis of the stability samples and

Storage effects due to freeze–thaw cycles, sample pretreatment, and reconstitution were also considered. For example, freeze–thaw cycles can affect the volatile and semi-volatile compound concentrations on a filter or in bulk dust. The pretreatment of samples before storage is performed when metabolism or reaction after collection is known to be of concern. For example, water samples for metals analysis are usually acidified to pH < 2 [86], and dust samples are sometimes irradiated to

Stability study samples with known concentrations of analytes were planned to be acquired in similar matrices to the NCS samples, i.e., particulate filters, wipes, and vacuum filters. These samples were to include (1) newly-prepared samples that have been spiked with known concentrations of analytes, and (2) pre-tested 'real-

Surface dust wipe and air diffusive samples could be made by spiking study sampling media and tap water samples by spiking analyte-free water with known amounts of the target analytes. These kind of samples can be purchased from reference material producers, proficiency testing sample providers, and/or accredited laboratories. To allow for a possible difference in reaction/degradation rate of analytes with their concentration in matrix, each matrix was to be spiked at environmentally relevant concentration levels, based on the literature review, and at concentrations that can be most reliably quantified by the analytical method employed, preferably at concentrations near the midpoint of the instrumental calibration range. Another benefit of this approach was that the concentration of the stability samples could be set to match the concentration used in the laboratory

Some samples cannot be spiked onto matrices in a manner representative of the corresponding environmental samples, or it would be very difficult to do so. There may be a difference in behavior of analytes in environmental samples and in similar spiked matrices due to pH, microbial enzymatic activity of the sample matrix, physicochemical properties of the analytes themselves and their reactions with the matrix, and other analytes present in the environment. For example, for vacuum filter and deposition plate stability samples, dust from non-participant homes could

Each spiked and real-world sample should represent the environmental sample matrix as close as possible to account for inter-analyte and analyte-matrix interactions. If it is not possible to include all analytes of interest in one matrix sample, they can be classified into groups by their chemical characteristics, and include representative analytes of each group deemed most unstable. Because sample homogeneity is very important, for example, surface wipe and dust samples should

**210**
