(1)
where [AngI]37 ° C represents the concentration of AngI after incubation at 37 °C, [AngI]blank represents the concentration of AngI after incubation on ice-water bath and Δt is the duration of the incubation.
In this method, the AngI is generated using a 3 h incubation at pH = 6 where renin is approximately twofold more enzymatically active than at physiological pH [5]. Generated AngI is extracted from the plasma samples using solid phase extraction (SPE) and analyzed by liquid chromatography and tandem mass spectrometry (LC-MS/MS ). The implicit assumption is that the endogenous angiotensinogen in the specimen will remain at a sufficiently high concentration in the 3 h incubation so as not to be rate-limiting. Proteolytic degradation of AngI by ACE and nonspecific peptidases is inhibited during AngI generation by both ethylenediaminetetraacetic acid (EDTA) and phenylmethanesulfonylfluoride (PMSF) which are present in the generation buffer.
Analytically, AngI was traditionally determined by radioimmunoassay (RIA), but with the advent of LC-MS/MS instruments into routine clinical laboratories, several authors have published methods suitable for high-throughput environments [6–10]. In addition to the benefit of avoiding radiotracers used in RIA, LC-MS/MS has a linear relationship between analyte concentration and instrument response. This affords a very wide analytical range thereby permitting the direct analysis of specimens from neonates (and from other high-renin states) without pre-dilution which causes unpredictable and patient-specific effects on recovery [5]. LC-MS/MS also permits the use of ion ratios to verify the absence of analytical interferents and, by virtue of its analytical sensitivity and specificity, likely obviates the need for blank subtraction, provided that the generation step is adequately long [8, 10].
2 Materials
2.1 Samples
2.2 Solvents and Reagents
1.
Bovine Serum Albumin , Fraction V, Omnipur® (EMD Millipore. Billerica, MA).
2.
Phenylmethanesulfonylfluoride (PMSF), >98.5 % (Sigma-Aldrich St. Louis, MO).
3.
100 mM PMSF in methanol: Dissolve 0.174 g of PMSF into 10 mL of methanol. Store at 2–8 °C. Expected stability: >6 months.
4.
Buffer A: 0.1 M Tris Base pH 6: Dissolve 12.11 g of Tris Base into 1000 mL of deionized water. Adjust to pH 6 with glacial acetic acid. Store at 2–8 °C. Expected stability: >6 months.
5.
Working Buffer B: 1 % BSA (w/v) in Stock Buffer A: Dissolve 0.1 g of BSA into 10 mL of 0.1 M Tris Buffer pH 6. Prepare fresh for each batch.
6.
Generation Buffer: Weigh 121.1 g of Tris Base + 74 g EDTA into a 1000 mL volumetric flask. Add DI water to about 900 mL. Sonicate for 30 min or until the Tris Base and EDTA are fully dissolved. Add DI water to the volume mark and mix well. Transfer to a labeled polypropylene storage container. Adjust to pH 5.45–5.50 with glacial acetic acid. Store at 2–8 °C. Expected stability: >6 months.
7.
On the day of analysis, add 100 μL of the 100 mM PMSF solution to 10 mL of generation buffer.
8.
Mobile Phase A: 0.2 % formic acid in water: Add 2 mL of formic acid to 1 L of DI water. Mix well.
9.
Mobile Phase B: 0.2 % formic acid in methanol: Add 2 mL of formic acid to 1 L of methanol. Mix well.
10.
10 % Formic Acid in water: Add 50 mL of formic acid to 450 mL of DI water. Mix well. Store at room temperature. Expected stability: >3 months.
11.
5 % formic acid in water: Add 25 mL of formic acid to 475 mL of DI water. Mix well. Store at room temperature. Expected stability: >3 months.
12.
20 % methanol in water: Add 100 mL of methanol to 400 mL of DI water. Mix well. Store at room temperature. Expected stability: >3 months.
13.
Lyphochek™ Hypertension Markers Control, Trilevel (Bio-Rad, Montreal QC, Canada).
2.3 Internal Standards and Standards
1.
Primary standard: AngI: 3× 10 μg (Proteochem, Loves Park, IL).
2.
Stable isotopically labeled internal standard (SIS): AngI (DRVYIHPFHL) with isotopically labeled arginine residue (13C,15N) was synthesized by the University of Victoria Genome BC Proteomics Centre (see Note 2 ).
3.
AngI stock solution (5000 ng/mL AngI in Working Buffer B): contents of 10 μg vials of AngI are dissolved in a total of exactly 2 mL of Working Buffer B to make a solution of 5000 ng/mL. Note that the dissolution process must be performed in 0.5 mL aliquots as the Proteochem vials are small in volume. Aliquot 500 μL of the 5000 ng/mL stock solution to microvials with lids, seal with parafilm, and store at −70 °C.
4.
Angiotensin S7: Remove an aliquot of the 5000 ng/mL stock solution and allow it to thaw at room temperature. Label ten polystyrene 10× 100 mm tubes appropriately (i.e., AngI S7 100 ng/mL, Date of Preparation). Add 1.96 mL of Working Buffer B to each tube. Aliquot 40 μL of the 5000 ng/mL stock solution to each of the ten tubes. Cap, Mix, and store at −70 °C until use.
5.
AngI SIS Solutions:
(a)
Stock solution, 1 mg/mL: dissolve 1 mg of AngI-SIS in DI water. Mix well to dissolve.
(b)
Intermediate working solution, 10 μg/mL: dilute the 1 mg/mL Stock Solution 100-fold to a resulting concentration of 10 μg/mL: add 100 μL of the 1 mg/mL stock solution to 9.90 mL of DI water. Mix well. Aliquot 10× 1 mL to labeled cryovials and store at −70 °C.
(c)
Working solution, 10 ng/mL in 10 % formic acid: Remove a vial of the 10 μg/mL Ang1-SIS Intermediate Working Solution and allow it to thaw. Mix well. Aliquot 0.5 mL into 500 mL of 10 % formic acid. The resulting concentration is 10 ng/mL. Store at 2–8 °C.
2.4 Calibrators and Controls
1.
Controls used are Bio-Rad Hypertension controls Levels 1, 2, and 3. The target mean and standard deviations are set based on results of 20 analyses over 10 runs. Controls are run in duplicate with each analytical run.
2.
An in-house patient pool is prepared approximately once yearly by pooling discarded anonymized previously analyzed patient plasma samples. The patient pool target value is set as per the Bio-Rad QC. The patient pool is run in duplicate with each analytical run.
3.
Calibrators are prepared in-house using the AngI stock solution in 1 % BSA in Buffer A (Working Buffer B).
2.5 Analytical Equipment and Supplies
1.
Strata-X 33u Polymeric Reversed Phase 96-Well Plate, 60 mg/well (Phenomenex, Torrance, CA).
2.
Vacuum manifold or positive pressure manifold, installed in robotic liquid handler or manually controlled.
3.
2 mL 96 deep square well, V-bottom plates (Corning, Corning, NY).
4.
Silicone cap mats with PTFE barrier for square well plates (Microliter Analytical Supplies, Suwanee GA).
5.
2 mL Nunc® 96 DeepWell™ round-bottom well plates (Thermo Scientific, Waltham, MA).
6.
Nunc® cap mats for round bottom plates (Thermo Scientific, Waltham, MA).
7.
API-5000 or API-5500 QTRAP® triple quadrupole mass spectrometer (AB SCIEX, Concord, ON) or other mass spectrometer capable of reaching the required limit of detection, equipped with appropriate software (e.g., Analyst®).
8.
Shimadzu 20 AC LC System with pumps, column oven, degasser, autosampler.
9.
Analytical column: 4u Proteo 90 Å, 50 × 2.0 mm (Phenomenex, Torrance, CA).
10.
Guard column, C12, 4 × 2.0 mm (Phenomenex, Torrance, CA).
3 Methods
3.1 Stepwise Procedure
1.
AngI S7 (100 ng/mL) is thawed at room temperature.
2.
Prepare Working Buffer B.
3.
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Prepare calibration standards by hand or on robotic liquid handler (see Note 3 ). The calibration standards (see Note 4 ) are serial dilutions of S7 as follows:
(a)
S6: 420 μL of S7 is mixed with 980 μL of Working Buffer B—resulting concentration is 30.00 ng/mL.
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