Table S1. Intra-assay Precision and Accuracy
Run ID
1
2
3
4
5
6
Theoretical Concentration
Mean
SD
%CV
%dev
n
Mean
SD
%CV
%dev
n
Mean
SD
%CV
%dev
n
Mean
SD
%CV
%dev
n
Mean
SD
%CV
%dev
n
Mean
SD
%CV
%dev
n
LLOQ
Extra Low
QC
Low QC
Middle QC
High QC
0.3125
0.3408
0.031
9.023
9.067
6
0.3173
0.030
9.390
1.547
6
0.3072
0.023
7.572
-1.707
6
0.3205
0.022
6.758
2.560
6
0.3295
0.019
5.722
5.440
6
0.3278
0.040
12.136
4.907
6
0.625
0.653
0.019
2.875
4.400
6
0.588
0.034
5.789
-5.947
6
0.628
0.018
2.798
0.453
6
0.642
0.019
2.912
2.773
6
0.634
0.066
10.430
1.360
6
0.643
0.021
3.247
2.907
6
1.500
1.593
0.097
6.067
6.167
6
1.551
0.064
4.141
3.389
6
1.638
0.099
6.074
9.200
6
1.585
0.135
8.495
5.633
6
1.404
0.114
8.124
-6.400
6
1.658
0.142
8.534
10.544
6
4.500
4.632
0.164
3.548
2.926
6
4.592
0.514
11.201
2.037
6
4.825
0.388
8.038
7.211
6
5.114
0.421
8.235
13.644
6
4.343
0.279
6.429
-3.493
6
4.878
0.268
5.496
8.389
6
18.000
17.219
1.045
6.068
-4.340
6
18.272
0.696
3.810
1.510
6
18.250
1.711
9.374
1.388
6
18.639
2.705
14.511
3.547
6
17.888
0.962
5.378
-0.621
6
19.011
1.832
9.635
5.615
6
Table S2. Inter-assay Precision and Accuracy
Theoretical Concentration
Mean Concentration
SD
%CV
%Dev
n
LLOQ
0.3125
0.3239
0.028
8.721
3.636
36
Extra Low
QC
0.625
0.631
0.038
6.022
0.991
36
Low QC
1.500
1.571
0.133
8.461
4.756
36
Midddle
QC
4.500
4.730
0.414
8.743
5.119
36
High QC
18.000
18.213
1.621
8.901
1.183
36
Table S3. Partial Volumes Assay
Dilution Factor
n
Theoretical Concentration
Mean Concentration
SD
%CV
%Dev
1:4
3
80
84.879
2.487
2.930
6.099
1:8
3
80
87.145
2.719
3.120
8.932
1:16
3
80
89.186
3.609
4.047
11.483
Table S4. Results of Stability Tests
STABILITY TEST
FREEZE/THAW 3 CYCLES
REINJECTION after 10 Days
AT 45ºC FOR 1 MONTH
AT 37ºC FOR 1 MONTH
AT ROOM TEMP. FOR 1 MONTH
AT 4ºC FOR 1 MONTH
AT -70ºC FOR 1 MONTH
a
STATISTICS
THEORETICAL CONC.
Mean
SD
%CV
%DEV from theoretical
%DEV from control
Mean
SD
%CV
%DEV from theoretical
%DEV from original run
Mean
SD
%CV
%DEV from theoretical
%DEV from control
Mean
SD
%CV
%DEV from theoretical
%DEV from control
Mean
SD
%CV
%DEV from theoretical
%DEV from control
Mean
SD
%CV
%DEV from theoretical
%DEV from control
Mean
SD
%CV
%DEV from theoretical
All studies were conducted in triplicate at each tested concentration
EFV CONCENTRATION (µg/mL)
Extra Low
Low QC
High QC
0.625
1.5
18
0.570
1.559
17.451
0.0759
0.2161
1.1694
13.3
13.9
6.7
-8.8
3.9
-3.1
-12.0
2.2
-9.5
0.647
1.542
18.563
0.0686
0.0801
1.6046
10.6
5.2
8.6
3.6
2.8
3.1
-0.6
2.8
3.1
0.671
1.418
17.767
0.0341
0.0871
1.1134
5.1
6.1
6.3
7.4
-5.5
-1.3
4.4
-14.5
-6.5
0.568
1.497
19.086
0.0308
0.0479
0.2399
5.4
3.2
1.3
-9.1
-0.2
6.0
-11.6
-9.7
0.4
0.646
1.680
19.292
0.0344
0.0884
0.9927
5.3
5.3
5.1
3.4
12.0
7.2
0.5
1.3
1.5
0.690
1.643
19.645
0.0401
0.0720
0.2639
5.8
4.4
1.3
10.4
9.6
9.1
7.3
-0.9
3.3
0.675
1.585
17.880
0.0153
0.0480
0.1661
2.3
3.0
0.9
8.1
5.7
-0.7
Table S5. Long-term stability of EFV DBS at -20 ºC
STABILITY TEST
1 WEEK
1 MONTH
3 MONTHS
6 MONTHS
1 YEAR
a
STATISTICS
THEORETICAL CONC.
Mean
SD
%CV
%DEV from theoretical
Mean
SD
%CV
%DEV from theoretical
Mean
SD
%CV
%DEV from theoretical
Mean
SD
%CV
%DEV from theoretical
Mean
SD
%CV
%DEV from theoretical
EFV CONCENTRATION (µg/mL)
Extra Low Low QC
High QC
0.625
1.5
18
0.588
1.551
18.272
0.034
0.064
0.696
5.8
4.1
3.8
-5.9
3.4
1.5
0.643
1.658
19.011
0.021
0.142
1.832
3.2
8.5
9.6
2.9
10.5
5.6
0.666
20.278
0.031
0.487
4.7
2.4
6.6
12.7
0.634
21.160
0.059
1.036
9.3
4.9
1.4
17.6
0.578
18.498
0.041
0.486
7.1
2.6
-7.6
2.8
All studies were conducted using 6 replicates at each tested concentration
Table S6. Assessing EFV Assay Specificity by Retention Time of Potential Concomitant Drugs
Retention time
Drug Name
abacavir
acyclovir
amprenavir
atazanvir
ciprofloxacin
clarithromycin
clindamycin
dapsone
darunavir
didanosine
diphenylhydramine
efavirenz
emtricitabine
etravirine
famotidine
fluoxetine
indinavir
itraconazole
lamivudine
lopinavir
miraviroc
naproxen
nelfinavir
nevirapine
phenytoin
prednisolone
rifampin
ritonavir
saquinavir
stavudine
sulfamethaxazole
tenofovir
thymidine
tipranavir
trimethropin
tylenol
zalcitabine
zidovudine
(min)
<3
<2.5
6.9
9
<5
<5
<5
<7
6.9
<2.5
<7
21
<3
No Peak
<7.5
<7.5
<5
<7.5
<3
18.1
<4
<7.5
<5
<3
<7
<7.5
<7
14.8
4.8
<2.5
<7.5
<3
<5
<5
<7.5
<5
<2.5
<3