Calibrator
Volume of standard stock solution (mL)
Volume of special reagent water (mL)
Final concentration (μmol/L)
1
1.0 (ADMA stock solution 2)
1.0 (SDMA stock solution 2)
2.0 (Arginine stock solution)
46.0
2.33 (ADMA)
4.42 (SDMA)
230 (Arginine)
2
0.5 (ADMA stock solution 2)
0.5 (SDMA stock solution 2)
1.0 (Arginine stock solution)
48.0
1.16 (ADMA)
2.21 (SDMA)
115 (Arginine)
3
1.0 (ADMA stock solution 3)
1.0 (SDMA stock solution 3)
0.5 (Arginine stock solution)
47.5
0.47 (ADMA)
1.32 (SDMA)
57.4 (Arginine)
4
0.5 (ADMA stock solution 3)
0.5 (SDMA stock solution 3)
0.5 (Arginine stock solution)
98.5
0.12 (ADMA)
0.33 (SDMA)
28.7 (Arginine)
5
0
50
0 (ADMA)
0 (SDMA)
0 (Arginine)
6.
HPLC /MS Check Stock Solution (34.9 μmol/L ADMA, 11.0 μmol/L SDMA, 2872 μmol/L Arginine in CLRW): Add 3 mL of ADMA standard stock solution 2, 0.5 mL of SDMA standard stock solution 2, and 5 mL of arginine standard stock solution to 10-mL volumetric flask, bring to volume with CLRW and mix well by inversion. Stable at −70 °C for 2 years.
7.
HPLC /MS Check Standard (0.55 μmol/L ADMA, 0.11 μmol/L SDMA, 28.7 μmol/L Arginine in CLRW): Add 0.5 mL of HPLC/MS Check stock solution to 50-mL volumetric flask, bring to volume with CLRW and mix well by inversion. Stable at −70 °C for 2 years.
2.4 Internal Standard and Quality Controls
1.
Primary internal standards (I.S.): asymmetric dimethylarginine hydrochloride (ADMA-d7) (C8H12ClD7N4O2 · H2O), 13C6-l-arginine hydrochloride (13C6H15ClN4O2) (Cambridge Isotope Laboratories).
2.
Quality Control Stock solutions: Primary standards are separately weighed or from different lots than those used to prepare calibrator stock solutions.
3.
ADMA-d7 I.S. Stock Solution (379 μmol/L primary I.S. in CLRW): Add 5 mg ADMA-d7 primary I.S. to 50-mL volumetric flask, bring to volume with CLRW and mix well by inversion. Stable at −70 °C for 2 years (see Note 1 ).
4.
13C6-Arginine I.S. Stock Solution (923 μmol/L primary I.S. in CLRW): Add 10 mg 13C6-arginine primary I.S. to 50-mL volumetric flask, bring to volume with CLRW and mix well by inversion. Stable at −70 °C for 2 years (see Note 1 ).
5.
I.S. Working Solution (1.90 μmol/L ADMA-d7, 36.9 μmol/L 13C6-Arginine in CLRW): Add 0.5 mL of ADMA-d7 I.S. stock solution and 4 mL of 13C6-Arginine I.S. stock solution to 100-mL volumetric flask, bring to volume with CLRW and mix well by inversion. Stable at −70 °C for 2 years (see Note 3 ).
6.
ADMA Quality Control Stock Solutions (7.27–3635 μmol/L primary standard in CLRW):
(a)
Add 10 mg ADMA primary standard to 10-mL volumetric flask, bring to volume with CLRW and mix well by inversion. This ADMA quality control stock solution 1 is 3635 μmol/L. Stable at −70 °C for 2 years (see Note 1 ).
(b)
Add 5 mL of previous ADMA quality control stock solution 1 to 50-mL volumetric flask, bring to volume with CLRW and mix well by inversion. This ADMA quality control stock solution 2 is 364 μmol/L. Stable at −70 °C for 2 years.
(c)
Add 2 mL of ADMA quality control stock solution 2 to 100-mL volumetric flask, bring to volume with CLRW and mix well by inversion. This ADMA quality control stock solution 3 is 7.27 μmol/L. Stable at –70 ºC for 2 years.
7.
SDMA Quality Control Stock Solutions (2.76–138 μmol/L primary standard in CLRW):
(a)
Add 10 mg SDMA primary standard to 100-mL volumetric flask, bring to volume with CLRW and mix well by inversion. This SDMA quality control stock solution 1 is 138 μmol/L. Stable at −70 °C for 2 years (see Note 1 ).
(b)
Add 2 mL of previous SDMA quality control stock solution 1 to 100-mL volumetric flask, bring to volume with CLRW and mix well by inversion. This SDMA quality control stock solution 2 is 2.27 μmol/L. Stable at −70 °C for 2 years.
8.
Arginine Quality Control Stock Solutions (2297–4594 μmol/L primary standard in CLRW).
(a)
Add 20 mg of arginine primary standard to 25 mL volumetric flask, bring to volume with CLRW and mix well by inversion. This arginine quality control stock solution 1 is 4594 μmol/L. Stable at −70 °C for 2 years (see Note 1 ).
(b)
Add 5 mL of the previous arginine quality control stock solution 1 to 10-mL volumetric flask, bring to volume with CLRW and mix well by inversion. This arginine quality control stock solution 2 is 2297 μg/mL. Stable at −70 °C for 2 years.
9.
Quality Controls (0.29–0.58 μmol/L ADMA, 0.33–0.55 μmol/L SDMA, 45.9–184 μmol/L, Arginine in charcoal stripped serum).
(a)
The levels of ADMA, SDMA, and arginine in the serum or plasma are predetermined by using this protocol or by an external laboratory using LC-MS/MS technology and are added to the final concentrations of the quality controls.
(b)
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Prepare low and high controls by diluting quality control stock solutions as shown in Table 2. For each dilution step: Add appropriate amount of quality control stock solution(s) for each ADMA, SDMA, and arginine as shown in Table 2 to 25-mL volumetric flask(s) and bring to volume with serum (or plasma ). Mix well by inversion after each dilution step. Stable at −70 °C for 2 years (see Notes 2 and 4 ).
Table 2
Preparation of quality controls
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