Download Development and validation of DBS steroid assay

Data Supplement
Development and validation of DBS steroid assay
Calibrators, controls and internal standards:
Calibrators:
Standards for androstenedione, 17-hydroxyprogesterone (17-OHP), and testosterone were obtained in
certified concentrations of 1 mg/mL. Dehydroepiandrosterone sulfate (DHEAS) was weighed as a solid
and diluted in methanol to a concentration of 1 mg/mL. A master steroid stock solution in methanol
was prepared containing final concentrations of 16,000 µmol/L DHEAS, 12,500 nmol/L Andro, 16,000
nmol/L Testo and 37,500 nmol/L 17-OHP. Steroid-free whole blood was used to make dilutions from the
steroid stock solution in order to give a six-point standard curve for each compound, spanning the target
ranges of 0.02 – 20 µmol/L for DHEAS, 0.04 – 40 nmol/L for androstenedione, 0.3 – 300 nmol/L for 17OHP and 0.1 – 70 nmol/L for testosterone. The resulting six whole blood calibrators were each spotted
onto a set of Whatman filter papers and allowed to dry. DBS calibrators were stored at -20°C until use.
After spotting the filter paper, the remaining whole blood calibrators were centrifuged and the plasma
was retrieved for analysis using the regular steroid assay being used clinically in the lab. The
concentration of the steroids measured in the plasma samples was determined by replicate analysis.
The values obtained for the calibrators from the plasma analysis were then used to set the calibrator
concentrations of the dried blood spot calibrators.
Controls
Controls were created in the same manner as the calibrators. Aliquots from the master steroid stock
solution were pipetted into whole blood pools created using washed red blood cells mixed with steroid
free serum. Two control levels were created with target concentrations of 0.3 and 16 µmol/L for DHEAS,
1.2 and 35 nmol/L for androstenedione, 15 and 275 nmol/L for 17-OHP and 5 and 65 nmol/L for
testosterone. The spiked pools were mixed gently, spotted onto filter paper, allowed to dry and then
stored at -20 C until use.
Internals standards:
All deuterated steroids were diluted to final concentrations of 17.35 nmol/L [d3]-testosterone, 150
nmol/L [d8]-17-hydroxyprogesterone, 17.45 nmol/L [d7]-androstenedione and 1.35 µmol/L [d2]-DHEAS.
Aliquots were stored at -80 C until use.
Assay Procedure and Validation
Methanol alone was found to extract the four steroid hormones together with the greatest overall
efficiency. Steroid recoveries are reported in Table S1. All reagents and samples were allowed to reach
room temperature prior to use. Six calibrators, a blank and two levels of controls were analyzed with
each run. Chromatography and mass spectrometry parameters for the un-derivatized assays are
presented in Table S2. Parameters for the derivatized assay are presented in Table S3.
LLOQ was defined and determined as the lowest measurable concentration of each steroid hormone
with acceptable imprecision (<20%). Recovery was determined as the average percent of the target
concentration recovered from DBS calibrators run as patient samples. Five samples were assayed at
each of three concentrations across the linear range for the recovery studies.
Imprecision studies were performed using the DBS controls, with 10 controls at each level assayed
within a single run for with-in run precision, and values at each control level from assays performed over
20 different days used for between-run precision.
Matrix effects and interferences had been previously studied with the validation of the serum steroid
assay and included assaying unextracted native steroids, extracted native steroids and native steroids
spiked into bovine serum albumin, serum, hemolyzed serum, lipemic serum and icteric serum and then
extracted. These studies showed no more than 15% loss or gain of concentration in any of the samples
assayed. In this DBS study, calibrators, controls and patient samples were all dried blood spots with the
same matrices. Thus for matrix effects, only different hemoglobin concentrations in the DBS were
studied.
In the serum assay, ion suppression was noted to be a problem affecting concentration when the
Internal Standard abundance of the patient samples was less than 60% of the average Internal Standard
abundance of the calibrators. For this reason, ion suppression was monitored throughout the DBS work
by determining that the IS abundance in DBS patient samples was always at least 80% of the average IS
abundance of the DBS calibrators.
DBS vs plasma
Paired plasma and DBS samples were prepared and analyzed as described in the main manuscript. 60
paired samples were analyzed for each steroid except DHEAS which only had 20 paired samples
analyzed. Samples with results below the LLOQ for the DBS or plasma steroid hormones were not used
to determine the relationship between DBS and plasma concentrations in patient samples.
RESULTS
The ions used to identify and quantify the steroid hormones are shown in Tables S2 and S3. Hormone
assays were linear from 0.3 - 40 nmol/L for androstenedione, 0.3 – 70 nmol/L for testosterone, 0.3 – 300
nmol/L for 17-OHP and 0.1 – 20 mol/L for DHEAS. The lower limit of quantification was defined as the
lowest value that could be measured with a coefficient of variation < 20%. Analytical measurement
range and LLOQ are shown in Table S4. The imprecision data for the assay are shown in Table S5 and
show that CVs ranged from 3.1 – 23.4% across the various steroid hormones and concentrations.
Table S1. Steroid recovery from DBS.
Analyte
17-OH-progesterone
Androstenedione
Testosterone
DHEAS
Percent recovery across linear range
98 – 112
99 – 113
99 – 107
92 – 101
Table S2. Instrument parameters for performing un-derivatized analysis
Time (min)
Buffer A (%)
0
100
1
100
6.1
10
6.2
0
8
0
8.1
100
9
100
Buffer A – 50% Methanol: 50% water
Buffer B – 100% Methanol
Column temperature = 35°C
Injection volume 20 μL
Flow rate – 0.4 mL/min
Compound
Androstenedione
Androstenedione
[d7]-Androstenedione
Testosterone
Testosterone
[d3]-Testosterone
17-hydroxyprogesterone
17-hydroxyprogesterone
[d8]-17hydroxyprogesterone
Dwell = 0.1 sec
MS1
(m/z)
287
287
294
289
289
293
331
331
339
Buffer B (%)
0
0
90
100
100
0
0
MS3
(m/z)
97
109
100
97
109
97
97
109
100
Retention
time (min)
3.16
Transition
Primary
Secondary
3.99
Primary
Secondary
4.44
Primary
Secondary
Cone
voltage
36 V
36 V
40 V
40 V
40 V
40 V
40 V
40 V
40 V
Collision
energy
24 eV
24 eV
24 eV
24 eV
24 eV
24 eV
24 eV
24 eV
24 eV
Table S3. Instrument parameters for performing un-derivatized analysis
Time (min)
Buffer A (%)
0
78
1
78
6
50
7
0
8
78
10
78
Buffer A – 0.1% formic acid in water
Buffer B – 0.1% formic acid in acetonitrile
Column temperature = 35°C
Injection volume 20 μL
Flow rate – 0.2 mL/min
Compound
DHEAS
DHEAS
[d2]-DHEA
Dwell = 0.1 sec
MS1
(m/z)
460
460
382
Buffer B (%)
22
22
50
100
22
22
MS3
(m/z)
362
95
96
Retention
time (min)
3.58
Transition
Primary
Secondary
Cone
voltage
48 V
48 V
50 V
Collision
energy
25 eV
35 eV
30 eV
Table S4. Linear range and LLOQ
Analyte
linear range
17-OH-progesterone
Androstenedione
Testosterone
DHEAS
LLOQ
0.30 – 300 nM
0.30 – 40 nM
0.30 – 70 nM
0.1 – 20 µM
concentration
CV (%)
0.3 nM
0.3 nM
0.3 nM
0.1 µM
15.7
13.8
12.9
16.6
Table S5. Imprecision studies.
Analyte
17-OH-progesterone
Androstenedione
Testosterone
DHEAS
Within run CVs (%)
Conc
CV
Conc
18 nM
4.2 260 nM
1.5 nM 13.3 32 nM
4.5 nM 12.3 64 nM
0.3 µM 13.3 16 µM
CV
4.9
8.0
8.1
3.1
Between run CVs (%)
conc
CV
conc
18 nM 16.9 260 nM
1.5 nM 23.2 32 nM
4.5 nM 23.4 64 nM
0.3 µM 21.1 16 µM
CV
6.2
10.4
9.4
10.4
Optimization of Blood Spot Extraction for Amino Acid Analysis
Preliminary experiments were performed to assess the utility of using multiple punches from one blood
spot because these spots are known to be heterogeneous from the center to the annulus. Three whole
blood samples were spotted onto standard Whatman 903 paper and residual sample centrifuged to
generate plasma. Spot concentrations were expressed as a percentage of the determined plasma value
to account for differences in endogenous amino acid concentrations. Figure S1 below shows spot
concentrations six representative amino acids determined using a one, three, or five punches from the
larger spot. Variability in spot concentrations using 3 punches was improved over a single spot with 5
punches yielding no additional benefit.
FIGURE S1.
Amino acid extraction efficiency.
A pool of EDTA anticoagulated whole blood was spotted onto Whatman 903 paper. An aliquot of the
same pool was supplemented with amino acid calibrator solutions to increase the concentration of each
amino acid by 75 µmol/L. Three punches from the native and supplemented pool from 10 spots each
were subject to extraction with methanol and recovery of added amino acid was calculated. Mean
percent recovery ± SD of the amino acids detectable in blood spots are presented below.
Table S6.
Amino Acid
Phenylalanine
Tyrosine
Isoleucine
Leucine
Valine
Threonine
Serine
Glycine
Methionine
Glutamine
Glutamate
Citrulline
Arginine
Ornithine
Homocitrulline
Alanine
Hydroxyproline
Proline
Lysine
β-aminoisobutyric
β-alanine
Sarcosine
γ-aminobutyrate
Histidine
α-amino-n-butyrate
Recovery, % (mean ±SD)
80±6
75±8
70±4
72±9
77±10
98±11
93±10
100±11
78±7
114±21
82±9
84±7
97±5
51±4
97±7
85±11
90±6
83±11
60±8
78±4
60±4
110±8
67±5
73±8
94±7