# Calculate Point Estimates of Fraction Unbound in Plasma (Fup) with Rapid Equilibrium Dialysis (RED) Data (Level-3) This function calculates the point estimates for the fraction unbound in plasma (Fup) using mass spectrometry (MS) peak areas from samples collected as part of **in vitro** measurements of chemical Fup using rapid equilibrium dialysis \insertCite waters2008validationinvitroTKstats. See the Details section for the equation(s) used in point estimation. ```r calc_fup_red_point( FILENAME, data.in, good.col = "Verified", output.res = FALSE, sig.figs = 3, INPUT.DIR = NULL, OUTPUT.DIR = NULL, verbose = TRUE ) ``` ## Arguments - `FILENAME`: (Character) A string used to identify the input level-2 file, "-fup-RED-Level2.tsv" (if importing from a .tsv file), and/or used to identify the output level-3 file, "-fup-RED-Level3.tsv" (if exporting). - `data.in`: (Data Frame) A level-2 data frame generated from the `format_fup_red` function with a verification column added by `sample_verification`. Complement with manual verification if needed. - `good.col`: (Character) Column name indicating which rows have been verified, data rows valid for analysis are indicated with a "Y". (Defaults to "Verified".) - `output.res`: (Logical) When set to `TRUE`, the result table (level-3) will be exported to the user's per-session temporary directory or `OUTPUT.DIR` (if specified) as a .tsv file. (Defaults to `FALSE`.) - `sig.figs`: (Numeric) The number of significant figures to round the exported result table (level-3). (Note: console print statements are also rounded to specified significant figures.) (Defaults to `3`.) - `INPUT.DIR`: (Character) Path to the directory where the input level-2 file exists. If `NULL`, looking for the input level-2 file in the current working directory. (Defaults to `NULL`.) - `OUTPUT.DIR`: (Character) Path to the directory to save the output file. If `NULL`, the output file will be saved to the user's per-session temporary directory or `INPUT.DIR` if specified. (Defaults to `NULL`.) - `verbose`: (**logical**) Indicate whether printed statements should be shown. (Default is TRUE.) ## Returns A level-3 data frame with one row per chemical, contains chemical identifiers such as preferred compound name, EPA's DSSTox Structure ID, calibration details, and point estimates for the fraction unbound in plasma (Fup) for all chemicals in the input data frame. ## Details The input to this function should be "level-2" data. Level-2 data is level-1, data formatted with the `format_fup_red` function, and curated with a verification column. "Y" in the verification column indicates the data row is valid for analysis. The data frame of observations should be annotated according to these types: |||||| |--:|--:|--:|--:|--:| |No Plasma Blank (no chemical, no plasma)|NoPlasma.Blank| |Plasma Blank (no chemical, just plasma)|Plasma.Blank| |Time zero chemical and plasma|T0| |Equilibrium chemical in phosphate-buffered well (no plasma)|PBS| |Equilibrium chemical in plasma well|Plasma| $f_{up}$ is calculated from MS responses as: c("$f_{up} = \\frac{\\max\\left( 0, \\frac{\\sum_{i=1}^{n_P} (r_P * c_{DF})}{n_P} - \\frac{\\sum_{i=1}^{n_{NPB}} (r_{NPB}*c_{DF})}{n_{NPB}}\\right)}\n$", "${\\frac{\\sum_{i=1}^{n_{PL}} (r_{PL} * c_{DF})}{n_{PL}} - \\frac{\\sum_{i=1}^{n_B} (r_B * c_{DF})}{n_B}}$") where $r_P$ is PBS Response, $n_P$ is the number of PBS Responses, $c_{DF}$ is the corresponding Dilution Factor, $r_{NPB}$ is No Plasma Blank Response, $n_{NPB}$ is the number of No Plasma Blank Responses, $r_{PL}$ is Plasma Response, $n_{PL}$ is the number of Plasma Responses, $r_{B}$ is Plasma Blank Response, and $n_B$ is the number of Plasma Blank Responses. If the output level-3 result table is chosen to be exported and an output directory is not specified, it will be exported to the user's R session temporary directory. This temporary directory is a per-session directory whose path can be found with the following code: `tempdir()`. For more details, see [https://www.collinberke.com/blog/posts/2023-10-24-til-temp-directories/](https://www.collinberke.com/blog/posts/2023-10-24-til-temp-directories/). As a best practice, `INPUT.DIR` (when importing a .tsv file) and/or `OUTPUT.DIR` should be specified to simplify the process of importing and exporting files. This practice ensures that the exported files can easily be found and will not be exported to a temporary directory. ## Examples ```r ## Load example level-2 data level2 <- invitroTKstats::fup_red_L2 ## scenario 1: ## input level-2 data from the R session and do not export the result table level3 <- calc_fup_red_point(data.in = level2, output.res = FALSE) ## scenario 2: ## import level-2 data from a 'tsv' file and export the result table ## Not run: ## Refer to sample_verification help file for how to export level-2 data to a directory. ## Unless a different path is specified in OUTPUT.DIR, ## the result table will be saved to the directory specified in INPUT.DIR. ## Will need to replace FILENAME and INPUT.DIR with name prefix and location of level-2 'tsv'. level3 <- calc_fup_red_point(# e.g. replace with "Examples" from "Examples-fup-RED-Level2.tsv" FILENAME="", INPUT.DIR = "", output.res = TRUE) ## End(Not run) ## scenario 3: ## import level-2 data from the R session and export the result table to the ## user's temporary directory ## Will need to replace FILENAME with desired level-2 filename prefix. ## Not run: level3 <- calc_fup_red_point(# e.g. replace with "MYDATA", FILENAME = "", data.in = level2, output.res = TRUE) # To delete, use the following code. For more details, see the link in the file.remove(list.files(tempdir(), full.names = TRUE, pattern = "-fup-RED-Level3.tsv")) ## End(Not run) ``` ## References \insertRef waters2008validationinvitroTKstats ## Author(s) John Wambaugh

calc_fup_red_point function