Last updated: 2025-04-03

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Summary

Cortisol value calculations

Min. 1st Qu. Median Mean 3rd Qu. Max. NA’s
A) Standard Method 0.4142 1.9653 7.5533 14.1906 28.0667 50.933 3
B) Spike-Corrected Method -45.870 -31.922 -1.929 -9.444 7.553 30.780 3
C) Standard, but simplified equation 0.4142 1.9653 7.5533 14.1906 28.0667 50.9333 3
D) Spike-Corrected (divided by two) -11.167 -4.193 2.349 5.314 17.368 30.780 3

Results:

  • Intra-assay CV: 14.5%

  • Intra-assay CV after removing low quality samples: 10%

  • Inter-assay CV: 21%

(Bindings for 20mg sample diluted in 250 uL, no spike: 64.8% and 48% in test3 and test4, respectively)

Conclusions:

Concerns: Overall quality of the plate is not great, but serial dilusions show clear parallelism and standards have values within the expected

Cortisol concentration calculations

# define volume of methanol used for cortisol extraction
# vol added / vol recovered (mL)
extraction <- 1/0.750

# set reading value of spike (std1, 0.333 ug/dL), 
# and transforming to ug.dL

std <- (3191+3228)/2
std.r <- (std/10000)
std.r
[1] 0.32095
# according to chatgpt, the spike's contribution is
# 1600 pg/mL, which is very similar to half of the reading for std 1 :]

Loading files and transforming units, including low quality data

df <- read.csv(file.path(data_path,"Data_QC_flagged.csv"))
kable(tail(df))
Wells Sample Category Weight_mg Buffer_nl Spike SpikeVol_uL Dilution Raw.OD Binding.Perc Conc_pg.ml Ave_Conc_pg.ml CV.Perc SD SEM CV_categ Binding.Perc_categ Failed_samples
77 G11 TP3A P 12 220 1 25 1 0.258 23.2 2800 2792 0.391 10.9 7.71 NA NA NA
78 H11 TP3A P 12 220 1 25 1 0.259 NA 2785 NA NA NA NA NA NA NA
79 A12 TP3B P 12 60 1 25 1 0.195 15.5 4084 4210 4.230 178.0 126.00 NA UNDER 20% binding UNDER 20% binding
80 B12 TP3B P 12 60 1 25 1 0.186 NA 4336 NA NA NA NA NA NA NA
81 C12 TP3C P 12 60 1 25 1 0.186 13.9 4336 4661 9.870 460.0 325.00 NA UNDER 20% binding UNDER 20% binding
82 D12 TP3C P 12 60 1 25 1 0.166 NA 4986 NA NA NA NA NA NA NA
# remove outlier
df<- df[(df$Sample != "TP3A"),]

# Creating variables in indicated units
# dilution (buffer)
df$Buffer_ml <- c(df$Buffer_nl/1000)

# remove unnecessary information 
data <- df %>%
    dplyr::select(Wells, Sample, Category, Binding.Perc, Ave_Conc_pg.ml, Weight_mg, Buffer_ml, Spike, SpikeVol_uL, Dilution, Failed_samples) 

kable(tail(data, 10))
Wells Sample Category Binding.Perc Ave_Conc_pg.ml Weight_mg Buffer_ml Spike SpikeVol_uL Dilution Failed_samples
71 A11 TP2A P 17.3 3793 9 0.06 1 25 1 UNDER 20% binding
72 B11 TP2A P NA NA 9 0.06 1 25 1 NA
73 C11 TP2B P 21.1 3101 9 0.06 1 25 1 NA
74 D11 TP2B P NA NA 9 0.06 1 25 1 NA
75 E11 TP2C P 18.1 3634 9 0.06 1 25 1 UNDER 20% binding
76 F11 TP2C P NA NA 9 0.06 1 25 1 NA
79 A12 TP3B P 15.5 4210 12 0.06 1 25 1 UNDER 20% binding
80 B12 TP3B P NA NA 12 0.06 1 25 1 NA
81 C12 TP3C P 13.9 4661 12 0.06 1 25 1 UNDER 20% binding
82 D12 TP3C P NA NA 12 0.06 1 25 1 NA
dim(data)
[1] 80 11
# remove duplicates
data <- data[!is.na(data$Binding.Perc), ]

(A) Standard Calculation

Formula:

((A/B) * (C/D) * E * 10,000) = F

  • A = μg/dl from assay output;
  • B = weight (in mg) of hair subjected to extraction;
  • C = vol. (in ml) of methanol added to the powdered hair;
  • D = vol. (in ml) of methanol recovered from the extract and subsequently dried down;
  • E = vol. (in ml) of assay buffer used to reconstitute the dried extract;
  • F = final value of hair CORT Concentration in pg/mg.
##################################
##### Calculate final values #####
##################################

# Transform to μg/dl from assay output

data$Ave_Conc_ug.dL <- c(data$Ave_Conc_pg.ml/10000)

data$Final_conc_pg.mg <- c(
    (data$Ave_Conc_ug.dL / data$Weight_mg) * # A/B *
      extraction *                                  # C/D  *     
      data$Buffer_ml * 10000)                 # E * 10000
data <- data[order(data$Sample),]
write.csv(data, file.path(data_path, "Data_cort_values_methodA.csv"), row.names = F)

# summary for all samples
summary(data$Final_conc_pg.mg)
   Min. 1st Qu.  Median    Mean 3rd Qu.    Max.    NA's 
 0.4142  1.9653  7.5533 14.1906 28.0667 50.9333       3 
kable(tail(data, 7))
Wells Sample Category Binding.Perc Ave_Conc_pg.ml Weight_mg Buffer_ml Spike SpikeVol_uL Dilution Failed_samples Ave_Conc_ug.dL Final_conc_pg.mg
67 E10 TP1B P 18.1 3820 6 0.06 1 25 1 HIGH CV;UNDER 20% binding 0.3820 50.93333
69 G10 TP1C P 27.8 2242 6 0.06 1 25 1 NA 0.2242 29.89333
71 A11 TP2A P 17.3 3793 9 0.06 1 25 1 UNDER 20% binding 0.3793 33.71556
73 C11 TP2B P 21.1 3101 9 0.06 1 25 1 NA 0.3101 27.56444
75 E11 TP2C P 18.1 3634 9 0.06 1 25 1 UNDER 20% binding 0.3634 32.30222
79 A12 TP3B P 15.5 4210 12 0.06 1 25 1 UNDER 20% binding 0.4210 28.06667
81 C12 TP3C P 13.9 4661 12 0.06 1 25 1 UNDER 20% binding 0.4661 31.07333
dim(data)
[1] 40 13

(B) Accounting for Spike

We followed the procedure described in Nist et al. 2020:

“Thus, after pipetting 25μL of standards and samples into the appropriate wells of the 96-well assay plate, we added 25μL of the 0.333ug/dL standard to all samples, resulting in a 1:2 dilution of samples. The remainder of the manufacturer’s protocol was unchanged. We analyzed the assay plate in a Powerwave plate reader (BioTek, Winooski, VT) at 450nm and subtracted background values from all assay wells. In the calculations, we subtracted the 0.333ug/dL standard reading from the sample readings. Samples that resulted in a negative number were considered nondetectable. We converted cortisol levels from ug/dL, as measured by the assay, to pg/mg—based on the mass of hair collected and analyzed using the following formula:

A/B * C/D * E * 10,000 * 2 = F

where - A = μg/dl from assay output; - B = weight (in mg) of collected hair; - C = vol. (in ml) of methanol added to the powdered hair; - D = vol. (in ml) of methanol recovered from the extract and subsequently dried down; - E = vol. (in ml) of assay buffer used to reconstitute the dried extract; 10,000 accounts for changes in metrics; 2 accounts for the dilution factor after addition of the spike; and - F = final value of hair cortisol concentration in pg/mg”

dSpike <- data

##################################
##### Calculate final values #####
##################################

dSpike$Final_conc_pg.mg <- 
  ifelse(
    dSpike$Spike == 1,    ## Only spiked samples
      ((dSpike$Ave_Conc_ug.dL - std.r) / # (A-spike) / B
      dSpike$Weight_mg) 
    * extraction *      # C / D
      dSpike$Buffer_ml * 10000 * 2 ,    # E * 10000 *2
    dSpike$Final_conc_pg.mg  
)

write.csv(dSpike, file.path(data_path, "Data_cort_values_methodB.csv"), row.names = F)

# summary for all samples
summary(dSpike$Final_conc_pg.mg)
   Min. 1st Qu.  Median    Mean 3rd Qu.    Max.    NA's 
-45.870 -31.922  -1.929  -9.444   7.553  30.780       3 
dSpikeSub <- data[c(data$Spike == 0), ]
summary(dSpikeSub$Final_conc_pg.mg)
   Min. 1st Qu.  Median    Mean 3rd Qu.    Max.    NA's 
 0.4142  0.8400  2.3493  7.7984 11.9733 30.7800       3 
kable(tail(dSpike, 10))
Wells Sample Category Binding.Perc Ave_Conc_pg.ml Weight_mg Buffer_ml Spike SpikeVol_uL Dilution Failed_samples Ave_Conc_ug.dL Final_conc_pg.mg
61 G9 TD6 D 99.7 81.99 20 0.11 1 0 0.031250 ABOVE 80% binding 0.008199 -45.870147
63 A10 TD7 D 99.0 86.73 20 0.11 1 0 0.015625 ABOVE 80% binding 0.008673 -45.800627
65 C10 TP1A P 21.8 2986.00 6 0.06 1 25 1.000000 NA 0.298600 -5.960000
67 E10 TP1B P 18.1 3820.00 6 0.06 1 25 1.000000 HIGH CV;UNDER 20% binding 0.382000 16.280000
69 G10 TP1C P 27.8 2242.00 6 0.06 1 25 1.000000 NA 0.224200 -25.800000
71 A11 TP2A P 17.3 3793.00 9 0.06 1 25 1.000000 UNDER 20% binding 0.379300 10.373333
73 C11 TP2B P 21.1 3101.00 9 0.06 1 25 1.000000 NA 0.310100 -1.928889
75 E11 TP2C P 18.1 3634.00 9 0.06 1 25 1.000000 UNDER 20% binding 0.363400 7.546667
79 A12 TP3B P 15.5 4210.00 12 0.06 1 25 1.000000 UNDER 20% binding 0.421000 13.340000
81 C12 TP3C P 13.9 4661.00 12 0.06 1 25 1.000000 UNDER 20% binding 0.466100 19.353333

(C) Skip unit transformation

##################################
##### Calculate final values #####
################################## 

data$Final_conc_pg.mg <- c(
    (data$Ave_Conc_pg.ml / data$Weight_mg) * # A/B *
      extraction *                                  # C/D  *     
      data$Buffer_ml)                 # E 
datac <- data[order(data$Sample),]
write.csv(datac, file.path(data_path, "Data_cort_values_methodC.csv"), row.names = F)

# summary for all samples
summary(datac$Final_conc_pg.mg)
   Min. 1st Qu.  Median    Mean 3rd Qu.    Max.    NA's 
 0.4142  1.9653  7.5533 14.1906 28.0667 50.9333       3 

(D) Account for Spike (contribution / 2)

Spike contribution (pg/mL) = (Vol. spike (mL) x Conc. spike (pg) ) / Vol. reconstitution (mL)

# Calculate contribution of spike according to the different volumes in which it was added
# considere that contribution in serial dilutions is smaller 

#datac$Spike.cont_pg.mL <-  datac

##################################
##### Calculate final values #####
##################################

dSpiked <- datac
dSpiked$Final_conc_pg.mg <- 
  ifelse(
    dSpike$Spike == 1,    ## Only spiked samples
      ((dSpike$Ave_Conc_pg.ml - (std/2)) / # (A-(spike/2)) / B
      dSpike$Weight_mg) 
    * extraction *      # C / D
      dSpike$Buffer_ml,    # E * 
    dSpike$Final_conc_pg.mg  
)

write.csv(dSpiked, file.path(data_path, "Data_cort_values_methodD.csv"), row.names = F)

# summary for all samples
summary(dSpiked$Final_conc_pg.mg)
   Min. 1st Qu.  Median    Mean 3rd Qu.    Max.    NA's 
-11.167  -4.193   2.349   5.314  17.368  30.780       3 

Plots

(A) Standard Calculation

# scatterplot method A
data$Spike <- replace(data$Spike, data$Spike == 1, 'Yes')
data$Spike <- replace(data$Spike, data$Spike == 0, 'No')
data$Buffer <- data$Buffer_ml
data$Buffer <- replace(data$Buffer, data$Buffer == 0.06, '60 uL')
data$Buffer <- replace(data$Buffer, data$Buffer == 0.11, '110 uL')
data$Buffer <- replace(data$Buffer, data$Buffer == 0.25, '250 uL')


ggplot(data, aes(y = Final_conc_pg.mg, 
                 x = Weight_mg, 
                 color = Spike,
                 shape = Buffer)) +
  geom_point(size = 2.5, alpha = 0.85) +  
   geom_text(aes(label = Sample), size = 2.5, vjust = -0.65, hjust = -0.18) +
    theme_minimal() +  
  geom_hline(yintercept = 0, 
             linetype = "dashed", color = "red") +
  xlim(0,52) +
  labs(
    title = "(A) Standard Calculation Cortisol Values",
    y = "Final Concentration (pg/mg)",
    x = "Weight (mg)") + 
  theme(
    plot.title = element_text(hjust = 0.5, 
                              size = 17, face = "bold"),
    axis.title = element_text(size = 14),  
    axis.text = element_text(size = 12)  
  ) + 
  scale_color_paletteer_d("vangogh::CafeTerrace")
Warning: Removed 3 rows containing missing values or values outside the scale range
(`geom_point()`).
Warning: Removed 3 rows containing missing values or values outside the scale range
(`geom_text()`).

(B) Accounting for Spike

dSpike$Spike <- replace(dSpike$Spike, dSpike$Spike == 1, 'Yes')
dSpike$Spike <- replace(dSpike$Spike, dSpike$Spike == 0, 'No')
dSpike$Buffer <- dSpike$Buffer_ml
dSpike$Buffer <- replace(dSpike$Buffer, dSpike$Buffer == 0.06, '60 uL')
dSpike$Buffer <- replace(dSpike$Buffer, dSpike$Buffer == 0.11, '110 uL')
dSpike$Buffer <- replace(dSpike$Buffer, dSpike$Buffer == 0.25, '250 uL')

# scatterplot
ggplot(dSpike, aes(y = Final_conc_pg.mg, 
                 x = Weight_mg, 
                 color = Spike,
                 shape = Buffer)) +
  geom_point(size = 3.5,  alpha = 0.85) +  
  geom_text(aes(label = Sample), size = 3, vjust = -1, hjust = -0.1) +
  theme_minimal() +  
  xlim(0,52) +
  geom_hline(yintercept = 0, 
             linetype = "dashed", color = "red") +
  labs(
    title = "(B) Calculation Accounting for Spike",
    y = "Final Concentration (pg/mg)",
    x = "Weight (mg)" ) +
  theme(
    plot.title = element_text(hjust = 0.5, 
                              size = 17, face = "bold"),
    axis.title = element_text(size = 14),  
    axis.text = element_text(size = 12)  
  )+ 
  scale_color_paletteer_d("vangogh::CafeTerrace")
Warning: Removed 3 rows containing missing values or values outside the scale range
(`geom_point()`).
Warning: Removed 3 rows containing missing values or values outside the scale range
(`geom_text()`).

(C)

# scatterplot method c
datac$Spike <- replace(datac$Spike, data$Spike == 1, 'Yes')
datac$Spike <- replace(datac$Spike, data$Spike == 0, 'No')
datac$Buffer <- data$Buffer_ml
datac$Buffer <- replace(datac$Buffer, data$Buffer == 0.06, '60 uL')
datac$Buffer <- replace(datac$Buffer, data$Buffer == 0.11, '110 uL')
datac$Buffer <- replace(datac$Buffer, data$Buffer == 0.25, '250 uL')


ggplot(datac, aes(y = Final_conc_pg.mg, 
                 x = Weight_mg, 
                 color = Spike,
                 shape = Buffer)) +
  geom_point(size = 2.5, alpha = 0.85) +  
   geom_text(aes(label = Sample), size = 2.5, vjust = -0.65, hjust = -0.18) +
    theme_minimal() +  
  geom_hline(yintercept = 0, 
             linetype = "dashed", color = "red") +
    #ylim(-26,24) +
#  xlim(0,52) +
  labs(
    title = "(C) ",
    y = "Final Concentration (pg/mg)",
    x = "Weight (mg)") + 
  theme(
    plot.title = element_text(hjust = 0.5, 
                              size = 17, face = "bold"),
    axis.title = element_text(size = 14),  
    axis.text = element_text(size = 12)  
  ) + 
  scale_color_paletteer_d("vangogh::CafeTerrace")
Warning: Removed 3 rows containing missing values or values outside the scale range
(`geom_point()`).
Warning: Removed 3 rows containing missing values or values outside the scale range
(`geom_text()`).

D

dSpiked$Spike <- replace(dSpiked$Spike, dSpiked$Spike == 1, 'Yes')
dSpiked$Spike <- replace(dSpiked$Spike, dSpiked$Spike == 0, 'No')
dSpiked$Buffer <- dSpiked$Buffer_ml
dSpiked$Buffer <- replace(dSpiked$Buffer, dSpiked$Buffer == 0.06, '60 uL')
dSpiked$Buffer <- replace(dSpiked$Buffer, dSpiked$Buffer == 0.11, '110 uL')
dSpiked$Buffer <- replace(dSpiked$Buffer, dSpiked$Buffer == 0.25, '250 uL')

# scatterplot
ggplot(dSpiked, aes(y = Final_conc_pg.mg, 
                 x = Weight_mg, 
                 color = Spike,
                 shape = Buffer)) +
  geom_point(size = 3.5,  alpha = 0.85) +  
  geom_text(aes(label = Sample), size = 3, vjust = -1, hjust = -0.1) +
  theme_minimal() +  
    ylim(-26,30) +
  xlim(0,52) +
  geom_hline(yintercept = 0, 
             linetype = "dashed", color = "red") +
  labs(
    title = "(D) ",
    y = "Final Concentration (pg/mg)",
    x = "Weight (mg)" ) +
  theme(
    plot.title = element_text(hjust = 0.5, 
                              size = 17, face = "bold"),
    axis.title = element_text(size = 14),  
    axis.text = element_text(size = 12)  
  )+ 
  scale_color_paletteer_d("vangogh::CafeTerrace")
Warning: Removed 4 rows containing missing values or values outside the scale range
(`geom_point()`).
Warning: Removed 4 rows containing missing values or values outside the scale range
(`geom_text()`).

Evaluation

# non-spiked samples only
data2 <-data

#two datasets, separated by dilution
data2.06 <- data2[data2$Buffer == "60 uL", ]
data2.11 <- data2[data2$Buffer == "110 uL", ]
data2.25 <- data2[data2$Buffer == "250 uL", ]


#### fit models ####

# model Buffer = 0.06
model06 <- lm(Final_conc_pg.mg ~ Weight_mg, 
              data = data2.06)
r_squared06 <- summary(model06)$r.squared

# model Buffer = 0.25
model25 <- lm(Final_conc_pg.mg ~ Weight_mg, 
              data = data2.25)
r_squared25 <- summary(model25)$r.squared

# Calculate residuals
residuals06 <- residuals(model06)
residuals25 <- residuals(model25)

# Quantify residuals
# Mean Absolute Error
mae06 <- mean(abs(residuals06))          
# Standard Deviation of Residuals
std_dev06 <- sd(residuals06)   

# Mean Absolute Error
mae25 <- mean(abs(residuals25))      
# Standard Deviation of Residuals
std_dev25 <- sd(residuals25)                      



# scatterplot

ggplot(data2, aes(y = Final_conc_pg.mg, 
                  x = Weight_mg, 
                  color = Category, 
                  fill = Category)) +
  geom_point(size = 2.5) +  
  geom_text(label = c(data2$Sample), nudge_y = 0.75, nudge_x = -0.5) +
  geom_smooth(method = "lm", 
              color = "gold3", 
              se = TRUE,
              alpha = 0.1) + 
  geom_hline(yintercept = mean(data2$Final_conc_pg.mg), 
             color = "gray80",
             linetype = "dashed") +
  geom_hline(yintercept = mean(data2.06$Final_conc_pg.mg), 
             color = "lightblue3",
             linetype = "dashed") +
  geom_hline(yintercept = mean(data2.25$Final_conc_pg.mg), 
             color = "lightpink3",
             linetype = "dashed") +
  theme_minimal() +  
  xlim(5, max(data2$Weight_mg) + 4) +
  ylim(0, max(data2$Final_conc_pg.mg)+4) + 
  labs(
    title = "Final Cort Concentration and Weight
    (Non-spiked only)",
    y = "Final Concentration (pg/mg)",
    x = "Weight (mg)"  
  ) +
  theme(
    plot.title = element_text(hjust = 0.5, size = 16, face = "bold"),  
    axis.title = element_text(size = 14),  
    axis.text = element_text(size = 12) 
  ) +
  # Add R^2 annotation
  annotate("text", x = max(data2$Weight_mg) * 0.7, 
           y = min(data2$Final_conc_pg.mg) * 1.5,
           label = paste("R² =", round(r_squared06, 3)), 
           size = 5, color = "black") +
  annotate("text", x = max(data2$Weight_mg) * 0.7, 
           y = max(data2$Final_conc_pg.mg) * 0.84,
           label = paste("R² =", round(r_squared25, 3)), 
           size = 5, color = "black")
Warning: Removed 3 rows containing non-finite outside the scale range
(`stat_smooth()`).
Warning: Removed 3 rows containing missing values or values outside the scale range
(`geom_point()`).
Warning: Removed 3 rows containing missing values or values outside the scale range
(`geom_text()`).
Warning: Removed 1 row containing missing values or values outside the scale range
(`geom_hline()`).
Removed 1 row containing missing values or values outside the scale range
(`geom_hline()`).
Removed 1 row containing missing values or values outside the scale range
(`geom_hline()`).
Warning: Removed 1 row containing missing values or values outside the scale range
(`geom_text()`).
Removed 1 row containing missing values or values outside the scale range
(`geom_text()`).

The previous figure shows that:

  • results are very stable across weights, particularly for the samples where a dilution of 250 uL was used
  • there is more error when using a dilution of 60 uL
  • dilution affects estimation of cortisol concentration in a significant way: even though final concentration numbers account for differences in the dilutions, the results we observe for each group do not overlap
  • the average value when using 250 uL of buffer is twice as big as when using 60 uL

Optimal dilution

Error using 0.06 mL buffer

Mean Absolute Error (MAE) 0.06 mL: 4.064 
Standard Deviation of Residuals 0.06 mL: 6.235 

Error using 0.25 mL buffer

Mean Absolute Error (MAE) 0.25 mL: 7.328 
Standard Deviation of Residuals 0.25 mL: 9.695 

From this we conclude that using a 250 uL dilution provides more consistent results


sessionInfo()
R version 4.4.3 (2025-02-28)
Platform: aarch64-apple-darwin20
Running under: macOS Sequoia 15.3.2

Matrix products: default
BLAS:   /Library/Frameworks/R.framework/Versions/4.4-arm64/Resources/lib/libRblas.0.dylib 
LAPACK: /Library/Frameworks/R.framework/Versions/4.4-arm64/Resources/lib/libRlapack.dylib;  LAPACK version 3.12.0

locale:
[1] en_US.UTF-8/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8

time zone: America/Detroit
tzcode source: internal

attached base packages:
[1] stats     graphics  grDevices utils     datasets  methods   base     

other attached packages:
[1] dplyr_1.1.4     paletteer_1.6.0 broom_1.0.7     ggplot2_3.5.1  
[5] knitr_1.49     

loaded via a namespace (and not attached):
 [1] sass_0.4.9        utf8_1.2.4        generics_0.1.3    tidyr_1.3.1      
 [5] prismatic_1.1.2   lattice_0.22-6    stringi_1.8.4     digest_0.6.37    
 [9] magrittr_2.0.3    evaluate_1.0.1    grid_4.4.3        fastmap_1.2.0    
[13] Matrix_1.7-2      rprojroot_2.0.4   workflowr_1.7.1   jsonlite_1.8.9   
[17] backports_1.5.0   rematch2_2.1.2    promises_1.3.0    mgcv_1.9-1       
[21] purrr_1.0.2       fansi_1.0.6       scales_1.3.0      jquerylib_0.1.4  
[25] cli_3.6.3         rlang_1.1.4       splines_4.4.3     munsell_0.5.1    
[29] withr_3.0.2       cachem_1.1.0      yaml_2.3.10       tools_4.4.3      
[33] colorspace_2.1-1  httpuv_1.6.15     vctrs_0.6.5       R6_2.5.1         
[37] lifecycle_1.0.4   git2r_0.35.0      stringr_1.5.1     fs_1.6.5         
[41] pkgconfig_2.0.3   pillar_1.9.0      bslib_0.8.0       later_1.3.2      
[45] gtable_0.3.6      glue_1.8.0        Rcpp_1.0.13-1     xfun_0.49        
[49] tibble_3.2.1      tidyselect_1.2.1  rstudioapi_0.17.1 farver_2.1.2     
[53] nlme_3.1-167      htmltools_0.5.8.1 rmarkdown_2.29    labeling_0.4.3   
[57] compiler_4.4.3